Analog of haemophilus HIN47 with reduced protease activity

ABSTRACT

An isolated and purified analog of Haemophilus influenzae Hin47 protein has a decreased protease activity which is less than about 10% of that of natural Hin47 protein and preferably substantially the same immunogenic properties as natural Hin47 protein. An isolated an purified nucleic acid molecule encoding the Hin47 analog may be provided in a recombinant plasmid which may be introduced into a cell which is grown to produce the Hin47 analog. Immunogenic compositions comprising the Hin47 analog and the encoding nucleic acid may be formulated as vaccines for in vivo administration to a host, including a human, to confer protection against diseases caused by a bacterial pathogen, including Haemophilus species, such as Haemophilus influenzae, that produces Hin47 protein or a protein capable of inducing antibodies in the host specifically reactive with Hin47 protein. The Hin47 analog and the encoding nucleic acid also may be employed in diagnostic applications.

REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/278,091 filed Jul. 21, 1994 (now U.S. Pat. No. 5,506,139).

FIELD OF THE INVENTION

The present invention relates to the field of immunology and isparticularly concerned with immunogens and antigens from species ofHaemophilus.

BACKGROUND TO THE INVENTION

Haemophilus influenzae is the organism responsible for a variety ofserious human diseases, such as meningitis, epiglotitis, pneumonia andotitis media. Haemophilus influenzae type b (Hib) is a major cause ofbacterial meningitis in children under the age of five years. Protectiveantibodies to the disease are induced by the capsular polysaccharide ofthe organism and vaccines have been developed that utilise the purifiedpolyribosyl ribitol phosphate (PRP) as the antigen. This vaccineprovides 90% protection in adults and in children over 24 months of age,but was ineffective in children under 24 months (Zangwill et al 1993).(The references are identified in a list of references at the end ofthis disclosure, each of which reference in the list is herebyincorporated by reference without further reference thereto). Like otherpolysaccharide antigens, PRP does not induce the proliferation ofT-helper cells, and re-immunisation fails to elicit either a boosterresponse or an increase in memory cells. Conjugation of the PRPpolysaccharide with protein carriers confers T-cell dependentcharacteristics to the vaccine and substantially enhances theimmunologic response to the PRP antigen. Currently, there are fourPRP-carrier conjugate vaccines available. These are vaccines based uponH. influenzae type b capsular polysaccharide conjugated to diphtheriatoxoid, tetanus toxoid, or Neisseria meningitidis outer membrane protein(reviewed in Zangwill et al, 1993). These H. influenzae b conjugatevaccines have dramatically reduced the incidence of bacterial meningitis(Schoendorf et al, 1994).

There are six serotypes of H. Influenzae designated a to f, which aredefined by their capsular polysaccharides. The current Haemophilusconjugate vaccines do not protect against other invasive typable strains(types a and c) and, importantly, do not protect against non-typable(NTHi) strains which are a common cause of postpartum and neonatalsepsis, pneumonia and otitis media. otitis media is the most commonillness of early childhood with approximately 70% of all childrensuffering at least one bout of otitis media before the age of seven.Chronic otitis media can lead to hearing, speech, and cognitiveimpairment in children. It is caused by bacterial infection withStreptococcus pneumoniae (approximately 50%), non-typable H. influenzae(approximately 30%), and Moraxella (Branhamella) catarrhalis(approximately 20%). In the United States alone, treatment of otitismedia costs between 1 and 2 billion dollars per year for antibiotics andsurgical procedures, such as tonsillectomies, adenoidectomies andinsertion of tympanostomy tubes. To achieve universal protection againstH. influenzae related diseases, particularly in the two to six month agegroup and certain high risk groups, the provision of conserved,cross-reactive non-capsular H. influenzae immunogens is desirable.Non-typable strains of H. influenzae are also important pathogensresponsible for pneumonia in the elderly and other individuals who areparticularly susceptible to respiratory infections. There is thus a needfor antigens from H. influenzae which are useful as components inimmunogenic preparations that provide protection against the manyserotypes of H. influenzae. PCT application WO 92/10936, published Jul.9, 1992 and incorporated herein by reference thereto, describes a 47,000molecular weight outer membrane protein obtained from H. influenzae thatis reported to be an adhesin and has been termed Hin47 that isimmunologically conserved between non-typable, type b and non-typedclinical isolates of H. influenzae. The amino acid sequence of Hin47 andthe nucleotide sequence of the gene encoding Hin47 were presented at theAmerican Society of Microbiology (ASM) conference held in New Orleans,May 26-30, 1992. These sequences have also been published in PCTapplication WO 94/00149, published Jan. 6, 1994 and incorporated hereinby reference thereto.

Since Hin47 is conserved among strains of Haemophilus influenzae, and isreported to be an adhesin, the protein has utility in diagnosis of andvaccination against disease caused by H. influenzae or other bacterialpathogens that produce Hin47 or a protein capable of raising antibodiesspecifically reactive with Hin47.

A disadvantage of Hin47 for use as an antigen in diagnosis, for thegeneration of anti-Hin47 antibodies useful in diagnosis and as animmunogen in vaccination is the unexpected discovery by the presentapplicants that Hin47 has protease activity which results in theautodigestion of Hin47 and the proteolytic degradation of other antigensmixed therewith.

It would be advantageous to provide analogs of Hin47 protein (sometimesreferred to herein as mutants or derivatives) that are substantiallyreduced in proteolytic activity for use as antigens, immunogenicpreparations including vaccines, carriers for other immunogens and thegeneration of diagnostic reagents.

SUMMARY OF THE INVENTION

The present invention is directed towards the provision of analogs ofHaemophilus Hin47 protein having reduced protease activity.

In accordance with one aspect of the invention there is provided anisolated and purified analog of Haemophilus influenzae Hin47 proteinhaving a decreased protease activity which is less than about 10% ofnatural Hin47 protein. Such Hin47 analog preferably has substantiallythe same immunogenic properties of natural Hin47 protein. The analog ofthe present invention may be produced by chemical, biochemical orgenetic modification of natural Hin47.

In one embodiment of the present invention, when the analog is producedby genetic modification, at least one amino acid of the natural Hin47contributing to protease activity may be deleted or replaced by adifferent amino acid to produce the reduced protease activity.Alternatively, the reduced protease activity may be achieved byinserting at least one amino acid into the natural Hin47 protein. The atleast one deleted or replaced amino acid may be selected from aminoacids 195 to 201 of Hin47, and specifically may be Serine-197, which maybe deleted or replaced by alanine. In addition, the at least one deletedor replaced amino acid may be His-91 and may be deleted or replaced byalanine or lysine or arginine. Further, the at least one deleted orreplaced amino acid may be Asp-121 and may be deleted or replaced byalanine or glutamic acid.

In a further aspect, the present invention provides an isolated andpurified nucleic acid molecule comprising a mutant Haemophilusinfluenzae hin47 gene encoding an analog of Haemophilus influenzae Hin47protein having a reduced protease activity which is less than about 10%of natural Hin47 protein. The mutant hin47 gene may encode any of theHin47 analogs discussed above. The mutant gene preferably is formed bysite-directed mutagenesis of a wild-type hin47 gene. The nucleic acidmolecule may be contained in a recombinant plasmid adapted fortransformation of a host and may be plasmid DS-1011-1-1 (deposited onJul. 27, 1994 at American type Culture Collection under Accession No.75845. The invention also includes a transformed cell containing such arecombinant plasmid.

The present invention, in another aspect, includes a method forproducing an analog of Haemophilus influenzae Hin47 protein having areduced protease activity which is less than about 10% of natural Hin47protein, which comprises identifying at least one amino acid residue ofHin47 protein which contributes to protease activity thereof, effectingsite-directed mutagenesis of the hin47 gene to remove or replace anucleotide sequence encoding the at least one amino acid and to producea mutated hin47 gene, introducing the mutated hin47 gene into a cell toproduce a transformed cell and growing the transformed cell to producethe Hin47 analog. The at least one amino acid which is selected may beany of the ones specifically identified above with respect to the Hin47analog.

The introduction of the mutated hin47 gene preferably produces atransformed cell in which the mutated hin47 gene is under control of theT7 promoter and the growing of the transformed cell and expression ofthe Hin47 analog by the T7 promoter then preferably is effected byculturing in an inducing concentration of lactose. Preferably, theintroduction of the mutated hin47 is effected by transforming the cellwith the recombinant plasmid DS-1011-1-1, sometimes otherwise referredto as plasmid pT7/Hin47*.

A further aspect of the invention provides a method of providingisolated and purified Hin47 analog, which comprises effecting theprocedure described above for the production of the Hin47 analog toproduce grown transformed cells harbouring inclusion bodies containingthe Hin47 analog, disrupting the grown transformed cells to producesupernatant and the inclusion bodies, solubilizing the inclusion bodiesto produce a solution containing Hin47 analog, chromatographicallypurifying the Hin47 analog from the solution free from cell debris, andisolating the purified Hin47 analog.

The analogs of Hin47 provided herein with their decreased proteolyticactivity are useful as antigens in immunogenic composition, carriers forother immunogens, diagnostic agents and in the generation of diagnosticagents. The nucleic acid molecules also are useful as probes fordiagnostic use and also as in immunogenic compositions.

In a further aspect of the invention, there is provided an immunogeniccomposition comprising an immuno-effective amount of the Hin47 analog orof the nucleic acid molecule including the gene encoding the Hin47analog. The immunogenic composition may be formulated as a vaccine forin vivo administration to a host, including a human, to conferprotection against diseases caused by a bacterial pathogen that producesHin47 or a protein capable of inducing antibodies in the hostspecifically reactive with Hin47. The bacterial pathogen may be aHaemophilus species, such as Haemophilus influenzae. The immunogeniccompositions of the invention may further comprise at least one otherimmunogenic or immunostimulating material, such as an adjuvant. In anadditional embodiment, the nucleic acid molecule comprising a geneencoding the Hin47 analog may be contained within a live vector, such asa pox virus, Salmonella, poliovirus, adenovirus, vaccinia or BCG.

The invention also extends to a method of generating an immune responsein a host, including a human, comprising administering thereto animmuno-effective amount of the immunogenic compositions provided herein.

As mentioned above, the Hin47 analog provided herein is useful indiagnostic applications. Accordingly, in an additional aspect of theinvention, there is provided a method of determining the presence ofantibodies specifically reactive with Hin47 in a sample, comprising thesteps of:

(a) contacting the sample with the Hin47 analog having substantially thesame immunogenic properties as the natural Hin47 protein as providedherein to produce complexes comprising the Hin47 analog and any suchantibodies present in the sample specifically reactive therewith; and

(b) determining production of the complexes.

The present invention also provides a method of determining the presenceof Hin47 in a sample, comprising the steps of:

(a) immunizing a subject with an immunogenic composition as providedherein to produce antibodies specific for Hin47 protein;

(b) contacting the sample with the antibodies to produce complexescomprising any Hin47 present in the sample and the Hin47 specificantibodies; and

(c) determining production of the complexes.

The invention also extends to a diagnostic kit for determining thepresence of antibodies in a sample specifically reactive with Hin47,comprising:

(a) the Hin47 analog having substantially the same immunogenicproperties as the natural Hin47 protein as provided herein;

(b) means for contacting the analog with the sample to produce a complexcomprising the analog and any such antibodies present in the sample; and

(c) means for determining production of the complex.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the restriction maps of plasmids JB-1031-1-14 andJB-1068-2-2 and the construction of the plasmids for sequence analysis;

FIGS. 2A, 2B, 2C, 2D, 2E, 2F, 2G and 2H show the full nucleotide (SEQ IDNO: 1) and deduced amino acid sequence (SEQ ID NO: 2) of Hin47 from H.influenzae strain SB33 as well as a partial nucleotide sequence (SEQ IDNO: 3) and a partial deduced amino acid sequence (SEQ ID No: 4) thereof,the latter being specifically copied by an inventor herein frommaterials presented in the ASM conference as described above;

FIGS. 3A and 3B show a comparison of the amino acid sequences of H.influenzae Hin47 (SEQ ID NO:2), E. coli htrA (SEQ ID NO: 5), andSalmonella typhimurium htrA (SEQ ID NO:6);

FIGS. 4A, 4B, 4C, 4D and 4E show an alignment of amino acid residues 57to 256 of Hin47 with certain known proteases (SEQ ID NOS: 7 to 16).Codes are as follows: TON, rat tonin; PKAAB, kallikrein; PTN, trypsin;CHAA, chymotrypsin; EST, elastase: RP2A, rat mast cell protease; SGT,Streptomyces griseus trypsin; SGBE, S.griseus proteinase A; SGA,S.griseus proteinase B; ALP, L.enzymogenes alpha-lytic protease; hin47,res. 57-256 of Hin47. Asterisks(*) denote structurally conservedregions. The catalytic triad residues are indicated by a hash mark (#).`con` refers to regions of structural concensus, among the mammalianproteases;

FIGS. 5A and 5B show the restriction maps for plasmids DS-1011-1-1 andDS-1048-2 which express a Hin47 analog from E. coli and a constructionscheme for plasmid DS-1011-1-1 (plasmid pT7/Hin47*);

FIG. 6, comprising panels A and B, shows a process for purifying theHin47 analog from E. coli according to one embodiment of the presentinvention (panel A) and gel analysis (panel B) of the purified product;

FIG. 7, comprising panels A, B and C, shows the protease activities ofnatural Hin47 and Hin47 analog towards β-casein;

FIG. 8, comprising panels A, B and C, shows the stability of naturalHin47 and the Hin47 analog at different temperatures;

FIG. 9, comprising panels A, B and C, shows the enzymatic degradation ofan H. influenzae recombinant protein by natural Hin47 and the Hin47analog; and

FIG. 10, comprising panels A, B and C, shows the comparativeimmunogenicity of natural Hin47 and the Hin47 analog in mice.

GENERAL DESCRIPTION OF INVENTION

Any Haemophilus strains that have Hin47 genes may be conveniently usedto provide the purified and isolated nucleic acid molecules (which maybe in the form of DNA molecules), comprising at least a portion codingfor Hin47 as typified by embodiments of the present invention. Suchstrains are generally available from clinical sources and from bacterialculture collections, such as the American Type Culture collection. Suchstrains include H. influenzae strains and other bacteria that produce aprotein capable of generating antibodies that specifically recognizeHin47 fragment or analog thereof. Appropriate strains of Haemophilus mayinclude:

H. influenzae type b strain MinnA;

H. influenzae type b strain Eagan;

H. influenzae non-typable strain SB33; or

H. influenzae non-typable strain PAK 12085.

Referring to FIG. 1, there is illustrated restriction maps of plasmidsJB-1031-1-14 and JB-1068-2-2 that contain a portion encoding Hin47protein from non-typable H. influenzae SB33. The nucleotide sequence ofthe Hin47 gene was determined and is shown in FIG. 2 along with thededuced amino acid sequence of the Hin47 protein. Referring to FIG. 3,there is shown an amino acid sequence alignment of H. Influenzae Hin47and the serine proteases htrA from Escherichia coli and htrA fromSalmonella typhimurium. This alignment for the first time reveals theunexpected discovery of the present applicants that Hin47 is related tobacterial serine proteases and that Hin47 has protease activity. Hin47has previously been reported to be an adhesin. The discovered proteaseactivity thereof greatly limits the usefulness of natural Hin47 as animmunogen for vaccination and as an antigen in diagnostic uses. Thesequence alignment shown in FIG. 3 revealed that the htrA proteins andHin47 contain a GNSGGAL (SEQ ID NO: 17) sequence between residues 195and 201 of the mature protein. The consensus sequence of the active siteof serine proteases is GDSGGPK (SEQ ID NO: 18) (Brenner, 1988) and theactive residue is serine. Thus, Serine-197 in Hin47 was mutated toproduce an analog of Hin47 reduced in protease activity, in accordancewith one embodiment of the invention. In a particular embodiment,Serine-197 was replaced by alanine. Amino acid residues 57 to 256 ofHin47 were further aligned with known proteases and the active siteresidues identified from the local homologies surrounding the residuesof the catalytic triad (FIG. 4). There is a standard numbering systemfor serine proteases in which the catalytic triad residues are numberedas His-57, Asp-102 and Ser-195. These correspond to residues His-91,Asp-121 and Ser-197 in the sequential numbering system. Thus, referringto FIG. 4, there is shown a structure-based alignment of tenstructurally determined serine proteases (SEQ ID NOS: 7 to 16) in whichhomologous residues are aligned primarily on the basis of similarlocations in three-dimensional space. The location of many of theresidues in the hydrophobic core of Hin47, as well as residues aroundthe active site can be aligned reasonably well to identify functionalamino acids of the Hin47 protease. Thus, other amino acid residues inHin47 that contribute to protease activity of the protein include His-91and Asp-121. In particular embodiments, His-91 may be replaced byalanine, lysine or arginine. In an additional embodiment, Asp-121 may bereplaced by alanine or glutamic acid. Although the provision of ananalog of Hin47 having reduced protease activity has been exemplifiedherein by particular amino acid substitution within Hin47 protein, thediscovery of the protease activity and the methods of Hin47 expression,purification and analysis provided herein, allow for the production ofother analogs having at least one other amino acid deleted or replacedor having at least one additional amino acid inserted into the Hin47protein. In particular applications and embodiments, it may be desirableto simultaneously alter several amino acids of the Hin47 protein toparticularly reduce the protease activity of Hin47. Accordingly, thepresent invention provides analogs of Hin47 protein having decreasedprotease activity due to single or multiple amino acid deletions,replacements or additions within the Hin47 protein.

Referring to FIG. 5, there is illustrated plasmids DS-1011-1-1 andDS-1048-2 which express a Hin47 analog serine-197→alanine in E. coli.FIG. 6 shows a flow diagram of a method for the purification of theHin47 analog from E. coli inclusion bodies.

FIG. 7 shows the reduced protease activity of the Hin47serine-197→alanine analog on the substrate β-casein and demonstrates theanalog to have less than about 10% of the protease activity of naturalHin47 protein. Thus, in one embodiment of the invention, there isprovided an analog of Hin47 having a protease activity of less thanabout 10% of the protease activity of natural Hin47 and such analog mayspecifically have amino acid Serine-197 replaced by alanine.

Referring to FIG. 8, there is illustrated an analysis of the increasedstability of an analog of Hin47 as provided herein. Thus, in oneembodiment of the present invention, there is provided an analog ofHin47 protein having increased thermal stability, and such analog mayspecifically have amino acid serine-197 replaced by alanine.

Referring to FIG. 9, there is illustrated the proteolytic degradation ofa non-Hin47 Haemophilus antigen by Hin47 and a Hin47 analog as providedherein. Thus, in accordance with a further embodiment of the presentinvention, there is provided an analog of Hin47 compatible with a secondnon-Hin47 protein and such analog may specifically have amino acidSerine-197 replaced by alanine.

Referring to FIG. 10 and Table 1, there is illustrated the comparativeimmunogenicity of unmodified Hin47 and a Hin47 analog having reducedprotease activity in mice. The Hin47 protein and Hin47 analog hadcomparable immunogenicity. Thus, in a particular embodiment, there isprovided an analog of Hin47 having reduced protease activity and havingsubstantially the same immunogenic properties of natural Hin47 protein.Such analog may specifically have amino acid Serine-197 replaced byalanine.

Referring to Table 2, there is shown the immunoprotective properties ofan analog of Hin47 having reduced protease activity against Hib in theinfant rat model of bacteraemia, according to an embodiment of theinvention and such analog may specifically have amino acid Serine-197replaced by alanine.

In accordance with another aspect of the present invention, there isprovided a vaccine against Haemophilus or other bacterial pathogens thatproduce Hin47 or a protein capable of inducing antibodies thatspecifically recognize Hin47, comprising an immunogenically-effectiveamount of an immunoprotective analog of Hin47 as provided herein or anucleic acid molecule having a sequence encoding a Hin47 analog asprovided herein, and a physiologically-acceptable carrier therefor. Theprovided analogs also may be used as a carrier protein for hapten,polysaccharides or peptides to make a conjugate vaccine againstantigenic determinants unrelated to Hin47.

As will be apparent from the following disclosure, the present inventionfurther provides plasmids and novel strains of bacteria for productionof Hin47 analogs as provided herein.

The purified and isolated DNA molecules comprising at least a portioncoding for an analog of Haemophilus influenzae Hin47 protein havingreduced protease activity compared to natural Hin47 typified by theembodiments described herein, are advantageous as nucleic acid probesfor the specific identification of Haemophilus strains in vitro or invivo. The Hin47 analogs encoded by the DNA molecules provided herein areuseful as diagnostic reagents as antigens or for the generation ofanti-Hin47 antibodies, antigens for the vaccination against the diseasescaused by species of Haemophilus and other bacterial pathogens thatproduce a protein capable of producing antibodies that specificallyrecognise Hin47 and for detecting infection by Haemophilus and othersuch bacteria.

In additional embodiments of the present invention, the Hin47 analogshaving reduced protease activity as provided herein may be used ascarrier molecules to prepare chimeric molecules and conjugate vaccines(including glycoconjugates) against pathogenic bacteria, includingencapsulated bacteria. Thus, for example, glycoconjugates of the presentinventions may be applied to vaccinations to confer protection againstdisease and infection caused by any bacteria having polysaccharideantigens including lipooligosaccharides (LOS) and PRP. Bacterialpathogens may include, for example, Haemophilus influenzae,Streptococcus pneumoniae, Escherichia coli, Neisseria meningitidis,Salmonella typhi, Streptococcus mutans, Cryptococcus neoformans,Klebsiella, Staphylococcus aureus and Pseudomonas aeruginosa. Particularantigens which can be conjugated to analogs of Hin47 and methods toachieve such conjugations are described in applicants published PCTapplication WO 94/12641 which is hereby incorporated by referencethereto.

In another embodiment, the carrier function of Hin47 analogs may beused, for example, to induce immunity toward abnormal polysaccharides oftumor cells, or to produce anti-tumor antibodies that can be conjugatedto chemotherapeutic or bioactive agents.

Accordingly, the present invention provides the primary sequence and thepreparation of an analog of Hin47 of H. influenzae that can be used inthe prevention and diagnosis of diseases caused by H. influenzae. Inparticular, the inventors discovered that the Hin47 analog can elicitprotective immune responses against live H. influenzae type b bacterialchallenge. Thus, the present inventions have utility in vaccines. Theinvention also discloses the nucleotide sequences of the gene encodingthe Hin47 analog. These DNA segments may be used to provide an immunogenessentially free from other H. influenzae antigens, such as PRP andlipooligosaccharides (LOS), through the application of recombinant DNAtechnology. The Hin47 analog protein, may be produced in a suitableexpression system, such as E. coli, Haemophilus, Bacillus, BordetellaFungi, Yeast, Baculovirus, Poxvirus, vaccinia or mammalian expressionsystems. The present disclosure further provides novel techniques whichcan be employed for preparing essentially pure Hin47 analogs.

It is clearly apparent to one skilled in the art, that the variousembodiments of the present invention have many applications in thefields of vaccination, diagnosis, treatment of, for example, Haemophilusinfections, and infections with other bacterial pathogens that produceproteins capable of producing antibodies that specifically recognizeHin47 and the generation of immunological reagents. A furthernon-limiting discussion of such uses is further presented below.

1. Vaccine Preparation and Use

Immunogenic compositions, suitable to be used as vaccines, may beprepared from Hin47 analogs as disclosed herein. The vaccine elicits animmune response in a subject which produces antibodies, includinganti-Hin47 antibodies and antibodies that are opsonizing orbactericidal. Should the vaccinated subject be challenged by Haemophilusor other bacteria that produce proteins capable of producing antibodiesthat specifically recognize Hin47, the antibodies bind to and inactivatethe bacterium. Furthermore, opsonizing or bactericidal anti-Hin47antibodies may also provide protection by alternative mechanisms.

Immunogenic compositions including vaccines may be prepared asinjectables, as liquid solutions or emulsions. The Hin47 analogs may bemixed with pharmaceutically acceptable excipients which are compatiblewith the Hin47 analog. Such excipients may include, water, saline,dextrose, glycerol, ethanol, and combinations thereof. The immunogeniccompositions and vaccines may further contain auxiliary substances, suchas wetting or emulsifying agents, pH buffering agents, or adjuvants toenhance the effectiveness thereof. Methods of achieving adjuvant effectinclude the use of agents such as aluminum hydroxide or phosphate(alum), commonly used as 0.05 to 0.1 percent solution in phosphatebuffered saline. Immunogenic compositions and vaccines may beadministered parenterally, by injection subcutaneously orintramuscularly. Alternatively, the immunogenic compositions formedaccording to the present invention, may be formulated and delivered in amanner to evoke an immune response at mucosal surfaces. Thus, theimmunogenic composition may be administered to mucosal surfaces by, forexample, the nasal or oral (intragastric) routes. Alternatively, othermodes of administration including suppositories and oral formulationsmay be desirable. For suppositories, binders and carriers may include,for example, polyalkalene glycols or triglycerides. Oral formulationsmay include normally employed incipients such as, for example,pharmaceutical grades of saccharine, cellulose and magnesium carbonate.These compositions can take the form of solutions, suspensions, tablets,pills, capsules, sustained release formulations or powders and containabout 1 to 95% of the Hin47 analogs. The immunogenic preparations andvaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective,protective and immunogenic. The quantity to be administered depends onthe subject to be treated, including, for example, the capacity of theindividual's immune system to synthesize antibodies, and if needed, toproduce a cell-mediated immune response. Precise amounts of activeingredient required to be administered depend on the judgment of thepractitioner. However, suitable dosage ranges are readily determinableby one skilled in the art and may be of the order of micrograms of theHin47 analogs. Suitable regimes for initial administration and boosterdoses are also variable, but may include an initial administrationfollowed by subsequent administrations. The dosage may also depend onthe route of administration and will vary according to the size of thehost.

The concentration of antigen in an immunogenic composition according tothe invention is in general about 1 to 95%. A vaccine which containsantigenic material of only one pathogen is a monovalent vaccine.Vaccines which contain antigenic material of several pathogens arecombined vaccines and also belong to the present invention. Suchcombined vaccines contain, for example, material from various pathogensor from various strains of the same pathogen, or from combinations ofvarious pathogens.

The nucleic acid molecules encoding the Hin47 analog of the presentinvention may also be used directly for immunization by administrationof the DNA directly, for example, by injection for genetic immunizationor by constructing a live vector, such as Salmonella, BCG, adenovirus,poxvirus, vaccinia or poliovirus. A discussion of some live vectors thathave been used to carry heterologous antigens to the immune system arediscussed in, for example, O'Hagan (1992). Processes for the directinjection of DNA into test subjects for genetic immunization aredescribed in, for example, Ulmer et al, 1993.

2. Immunoassays

The Hin47 analogs of the present invention are useful as immunogens forthe generation of anti-Hin47 antibodies, as antigens in immunoassaysincluding enzyme-linked immunosorbent assays (ELISA), RIAs and othernon-enzyme linked antibody binding assays or procedures known in the artfor the detection of anti-bacterial, Haemophilus, and anti-Hin47antibodies. In ELISA assays, the Hin47 analogs, are immobilized onto aselected surface, for example, a surface capable of binding proteinssuch as the wells of a polystyrene microtiter plate. After washing toremove incompletely adsorbed Hin47 analogs, a nonspecific protein suchas a solution of bovine serum albumin (BSA) that is known to beantigenically neutral with regard to the test sample may be bound to theselected surface. This allows for blocking of nonspecific adsorptionsites on the immobilizing surface and thus reduces the background causedby nonspecific bindings of antisera onto the surface.

The immobilizing surface is then contacted with a sample, such asclinical or biological materials, to be tested in a manner conducive toimmune complex (antigen/antibody) formation. This may include dilutingthe sample with diluents, such as solutions of BSA, bovine gammaglobulin (BGG) and/or phosphate buffered saline (PBS)/Tween. The sampleis then allowed to incubate for from 2 to 4 hours, at temperatures suchas of the order of about 25° to 37° C. Following incubation, thesample-contacted surface is washed to remove non-immunocomplexedmaterial. The washing procedure may include washing with a solution,such as PBS/Tween or a borate buffer. Following formation of specificimmunocomplexes between the test sample and the bound Hin47 analogs, andsubsequent washing, the occurrence, and even amount, of immunocomplexformation may be determined by subjecting the immunocomplex to a secondantibody having specificity for the first antibody. If the test sampleis of human origin, the second antibody is an antibody havingspecificity for human immunoglobulins and in general IgG. To providedetecting means, the second antibody may have an associated activitysuch as an enzymatic activity that will generate, for example, a colourdevelopment upon incubating with an appropriate chromogenic substrate.Quantification may then be achieved by measuring the degree of colourgeneration using, for example, a visible spectra spectrophotometer.

3. Use of Sequences as Hybridization Probes

The nucleic acid molecules of the present invention, having the sequenceof the hin47 analog gene, allow for the identification and cloning ofthe Hin47 genes from any species of Haemophilus and other bacteria thatproduce proteins capable of producing antibodies that specificallyrecognize Hin47.

The nucleic acid molecules having the sequence encoding the Hin47 analogof the present invention are useful for their ability to selectivelyform duplex molecules with complementary stretches of other hin47 genes.Depending on the application, a variety of hybridization conditions maybe employed to achieve varying degrees of selectivity of the probetoward the other hin47 genes. For a high degree of selectivity,relatively stringent conditions are used to form the duplexes, such aslow salt and/or high temperature conditions, such as provided by 0.02 Mto 0.15 M NaCl at temperatures of between about 50° to 70° C. For someapplications, less stringent hybridization conditions are required, suchas 0.15 M to 0.9 M salt, at temperatures ranging from between about 20°C. to 55° C. Hybridization conditions can also be rendered morestringent by the addition of increasing amounts of formamide, todestabilize the hybrid duplex. Thus, particular hybridization conditionscan be readily manipulated, and will generally be a method of choicedepending on the desired results.

In a clinical diagnostic embodiment, the nucleic acid molecules encodingthe hin47 genes of the present invention may be used in combination withan appropriate means, such as a label, for determining hybridization. Awide variety of appropriate indicator means are known in the art,including radioactive, enzymatic or other ligands, such asavidin/biotin, which are capable of providing a detectable signal. Insome diagnostic embodiments, an enzyme tag, such as urease, alkalinephosphatase or peroxidase, instead of a radioactive tag may be used. Inthe case of enzyme tags, calorimetric indicator substrates are knownwhich can be employed to provide a means visible to the human eye orspectrophotometrically, to identify specific hybridization with samplescontaining hin47 gene sequences.

The nucleic acid molecules comprising hin47 genes of the presentinvention are useful as hybridization probes in solution hybridizationsand in embodiments employing solid-phase procedures. In embodimentsinvolving solid-phase procedures, the test DNA (or RNA) from samples,such as clinical samples, including exudates, body fluids (e.g., serum,amniotic fluid, middle ear effusion, sputum, bronchoalveolar lavagefluid) or even tissues, is adsorbed or otherwise affixed to a selectedmatrix or surface. The fixed, single-stranded nucleic acid is thensubjected to specific hybridization with selected probes comprising thenucleic acid sequences of the hin47 genes of the present invention underdesired conditions. The selected conditions will depend on theparticular circumstances based on the particular criteria requireddepending on, for example, the G+C contents, type of target nucleicacid, source of nucleic acid, size of hybridization probe etc. Followingwashing of the hybridization surface so as to remove non-specificallybound probe molecules, specific hybridization is detected, or evenquantified, by means of the label.

4. Expression of the Genes Encoding Analogs of Hin47 Having ReducedProtease Activity

Vectors perhaps containing replicon and control sequences which arederived from species compatible with the host cell may be used for theexpression of the Hin47 analog genes as provided herein in expressionsystems. The vector ordinarily carries a replication site, as well asmarking sequences which are capable of providing phenotypic selection intransformed cells. For example, E. coli may be transformed using pBR322which contains genes for ampicillin and tetracycline resistance and thusprovides easy means for identifying transformed cells. The pBR322plasmid, or other microbial plasmid or phage must also contain, or bemodified to contain, promoters which can be used by the host cell forexpression of its own proteins.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host can be used as a transforming vectorin connection with these hosts. For example, the phage in lambda GEM™-11may be utilized in making recombinant phage vectors which can be used totransform host cells, such as E. coli LE392.

Promoters commonly used in recombinant DNA construction include theβ-lactamase (penicillinase) and lactose promoter systems (Chang et al,1979; Goeddel et al, 1980) and other microbial promoters, such as the T7promoter system (U.S. Pat. No. 4,952,496). Details concerning thenucleotide sequences of promoters are known, enabling a skilled workerto ligate them functionally with plasmid vectors. The particularpromoter used will generally be a matter of choice depending upon thedesired results. Hosts that are appropriate for expression of the Hin47analogs include E. coli Bacillus species, Haemophilus Bordetella fungi,yeast, mammalian cells or the baculovirus expression system may be used.

Thus, in accordance with the invention, it may be preferred to make theHin47 analog protein by recombinant methods. Particularly desirablehosts for expression in this regard include Gram positive bacteria whichdo not have LPS and are therefore endotoxin free. Such hosts includespecies of Bacillus and may be particularly useful for the production ofnon-pyrogenic Hin47 analog.

Biological Deposits

Plasmid DS-1011-1-1 (pT7/Hin47*) that contains a portion coding for aHin47 analog that is described and referred to herein has been depositedwith the American Type Culture Collection (ATCC) located at Rockville,Md., USA, pursuant to the Budapest Treaty and prior to the filing ofthis continuation-in-part application on Jul. 27, 1994 under AccessionNo. 75845. Samples of the deposited plasmid will become available to thepublic upon grant of a patent based upon this United States patentapplication. The invention described and claimed herein is not to belimited in scope by plasmid deposited, since the deposited embodiment isintended only as an illustration of the invention. Any equivalent orsimilar plasmids that encode similar or equivalent antigens as describedin this application are within the scope of the invention.

EXAMPLES

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitations.

Methods of molecular genetics, protein biochemistry, and immunology usedbut not explicitly described in this disclosure and these Examples areamply reported in the scientific literature and are well within theability of those skilled in the art.

Example 1

This Example illustrates the cloning of the hin47 gene from non-typableH. influenzae strain SB33.

Chromosomal DNA was prepared from H. influenzae strain SB33, and anEMBL3 library was prepared and screened with a labelled oligonucleotideprobe specific for the 5'-end of hin47. Non-typable H. influenzae strainSB33 was grown on Mueller-Hinton agar or in brain heart infusion brothas described by Harkness et al, 1992. Chromosomal DNA was prepared asfollows: cells from 50 ml of culture were pelleted by centrifugation at5000 rpm for 15 to 20 min, at 4° C., in a Sorvall RC-3B centrifuge. Thecell pellet was resuspended in 10 ml of TE (10 mM Tris/HCl, 1 mM EDTA,pH 7.5), pronase was added to 500 μg ml⁻¹ and SDS to 1%. The sample wasincubated at 37° C. until a clear lysate was obtained. The lysate wasgently extracted once with Tris-saturated phenol (pH 7.4), once withTris-saturated phenol/chloroform (1:1) and once with chloroform. Thefinal aqueous phase was dialysed at 4° C. for 24 h against 1M NaCl,followed by 24 h against TE.

An EMBL3 library was prepared by partial digestion of SB33 chromosomalDNA with Sau3A I, followed by size fractionation either on a 10 to 30%sucrose gradient in TNE (20 mM Tris/HCl, 5 mM NaCl, 1 mM EDTA, pH 8.0)or by preparative gel electrophoresis. Fractions containing DNAfragments greater than 5 kb in length were pooled, precipitated andligated with BamH I arms of EMBL3 (Promega). The ligation mixture waspackaged using a Gigapack II packaging kit and plated onto E. coli LE392cells. The libraries were amplified and stored at 4° C. in the presenceof 0.3% chloroform.

Plaques were lifted onto nitrocellulose filters for hybridization with a³² P-labelled oligonucleotide probe (3026.SL). The oligonucleotidesequence was ATGAAAAAAACACGTTTTGTATTAAATAGTATTGCACTTGG (SEQ ID NO: 3)corresponding to the N-terminal amino acid sequence MKKTRFVLNSIALG (SEQID NO: 19). Phage DNA was prepared from putative plaques and the insertDNA was excised by Sal I digestion and cloned into pUC8-BgXb digestedwith Sal I. Plasmids JB-1031-1-14 and JB-1068-2-2 (FIG. 1) were selectedfor further analysis.

Example 2

This Example illustrates the characterization and sequence analysis ofthe hin47 gene and deduced amino acid sequence of the Hin47 protein fromNTHi strain SB33.

Restriction mapping and Southern blot analysis of clones JB-1031-1-14and JB-1068-2-2 localized the hin47 gene on a 4.7 kb BamH I/BamH I or a2.7 kb BamH I/Pst I DNA fragment. The 4.7 kb BamH I/BamH I fragment fromJB-1068-2-2 was subcloned into pUC8/BgXb generating plasmid DS-755-1.The 3.1 kb BamH I to Xba I fragment of DS-755-1 was subcloned into pUC18generating plasmid JB-1165-1 which has restriction sites suitable forthe Erase-a-base (Promega) procedure (FIG. 1). This technique generatessuccessive clones with increasing truncations of insert DNA, with thedeletions occurring from the same end. The resultant nested set ofclones can be sequenced rapidly using a universal primer.

DNA from plasmid JB-1165-1 was digested with BamH I and Sac I andsubjected to exoIII digestion using an Erase-a-base kit. The resultantset of truncated plasmids was analysed by agarose gel electrophoresisand representative plasmids were selected for sequence analysis.

Plasmid DNA for sequencing was prepared by a modification of theprocedure of Holmes and Quigley, 1981. Briefly, the cell pellet from 50ml of culture was resuspended in 10 ml STET (8% sucrose, 5% TritonX-100, 50 mM EDTA, and 50 mM Tris/HCl, pH 8.0), lysozyme (2.5 mg) wasadded and the mixture was boiled for 2 min. The sample was spun at14,000 rpm in a Sorvall RC 5B for 20 minutes and the supernatant wasprecipitated with an equal volume of isopropanol, washed with 70%ethanol then absolute ethanol, and then air dried. The pellet wasresuspended in 0.9 ml of TE, then 20 μl of 5 mg ml⁻¹ RNAse A were added,and the mixture was incubated at 37° C. for 15 min. After the additionof 500 μl of 1.5M NaCl/30% PEG, the mixture was incubated on ice for 30min and the DNA was pelleted by centrifugation in an Eppendorf microfugefor 10 min. The pellet was resuspended in 400 μl of TE and extractedtwice with Tris-saturated phenol (pH 7.4), twice with Tris-saturatedphenol/chloroform (1:1) and twice with chloroform. The DNA wasprecipitated by adding 40 μl of 3M ammonium acetate and 1 ml of ethanol,washed with 70% ethanol and resuspended in distilled water.

DNA samples were sequenced using the ABI model 370A DNA sequencer andthe dye terminator chemistry. The universal reverse primer was used withthe nested set of clones to determine the sequence of the hin47 codingstrand. Oligonucleotide primers of approximately 25 bases in length wereused to confirm the sequence of the non-coding strand. The nucleotidesequence of the SB33 hin47 gene and the deduced amino acid sequence ofthe Hin47 protein are shown in FIG. 2. The nucleotide and N-terminalamino acid sequences of Hin47 presented at the ASM meeting, New Orleans,May 26 to 30, 1992 are indicated in lower case on FIG. 2. The aminoterminal sequences of the SB33 Hin47 and this presented sequence areidentical, establishing the identity of the cloned gene as hin47.

Example 3

This Example describes the discovery of serine protease activity ofHin47 protein.

The deduced amino acid sequence of Hin47 protein determined in Example 2above was compared with all other known proteins in the Genbank database. As described above, Hin47 protein is described in published PCTapplications WO 94/00149, WO 92/11367 and WO 92/10936 to be an adhesinmolecule of Haemophilus. It was, therefore, a surprising and unexpecteddiscovery of the present invention that Hin47 protein has significantamino acid homology (55%) with the serine proteases E. coli htrA and S.typhimurium htrA and other proteases. These amino acid sequencehomologies are shown in FIGS. 3 and 4. Furthermore, Hin47 protein wasfound to autodigest unless it was stored in the presence of a serineprotease inhibitor, such as Pefablock.

Example 4

This Example illustrates the generation of the mutant hin47 gene bysite-directed mutagenesis.

As explained above, H. influenzae Hin 47, E. coli htrA, and S.typhimurium htrA are all serine proteases. The consensus sequence of theactive site of serine proteases is GDSGGPK (SEQ ID NO: 18) Brenner,1988! with serine being the active residue. The htrA proteins both havea GNSGGAL (SEQ ID NO: 17) sequence and in H. influenzae Hin47, there isthe identical sequence between residues 195 and 201 of the matureprotein. Thus, the serine residue at position 197 was selected forsite-directed mutagenesis, to produce an analog of Hin47 with reducedprotease activity.

An oligonucleotide CGCTCCACCAGCATTACCGCGG (SEQ ID NO: 20) wassynthesized which would change the serine residue at 197 to an alanine.The hin47 gene was cloned into M13mp18 generating clone DS-981-3 andmutagenesis was performed using the Amersham In Vitro Site-DirectedMutagenesis kit. Clone DS-991-8 was confirmed by sequence analysis tocontain the mutation Serine-197 to Alanine. This mutant hin47 gene isdesignated hin47*.

In addition a comparison of the amino acid sequence of Hin47 with otherproteases (as shown in FIG. 4) revealed that amino acids His-91 andAsp-121 are sites appropriate for mutagenesis to produce an analog ofHin47 with reduced protease activity. By mutagenesis methods analogousto those described above, His-91 and/or Asp-121 are deleted or replacedby different amino acids. Such amino acid replacements may includeHis-91 to Alanine and Asp-121 to Alanine. Olignonucleotides to effectsuch mutagenesis include:

His-91→Ala-91 5' ATCAATAACAGCATTATTGGT 3' (SEQ ID NO: 21)

Asp-121→Ala-121 5' TAATGCAATTGCTGATAGTTC3' (SEQ ID NO: 22)

Many serine proteases are secreted in an inactive (`zymogen`) form, andrequire clipping to expose their active sites. N terminal sequenceanalysis of mature natural Hin47 protein suggested the cleavage of thepreprotein to occur at KFFFG DRFAEQ (SEQ ID NO: 23). Modifications ofamino acids that prevent cleavage of the molecule to produce the activeprotease molecule can produce an analog of Hin47 having reduced proteaseactivity.

Example 5

This Example illustrates the construction of plasmids expressing Hin47Ser-197→alanine analog from E. coli.

The mutated hin47* gene from plasmid DS-991-8 was cloned into the pT7-7expression vector to generate plasmid DS-1011-1-1 (FIG. 5). E. colistrain BL21/DE3 was transformed to generate E. coli strain DS-1018-3-1which expresses Hin47 Ser-197→alanine analog upon induction.

In order to utilize tetracycline selection, the hin47* gene was clonedinto pBR328. The Bgl II/Cla I T7/hin47* gene fragment from DS-1011-1-1was cloned into pEVvrf1 (Young and Davis, 1985) in order to generate aBgl II/BamH I fragment which could be cloned into pUC-4K (Pharmacia)digested with BamH I. The resultant clone DS-1034-3 was digested withEcoR I and the T7/hin47* gene fragment cloned into pBR328 (BoehringerMannheim Corporation) to generate plasmids DS-1048-2 and DS-1067-2.Electroporation of plasmid DNA into E. coli strain BL21/DE3 resulted instrains DS-1071-1-1 and DS-1071-3-1 which express the Hin47Ser-197→alanine analog.

Example 6

This Example illustrates the expression of Hin47 Ser-197→alanine analogfrom E. coli.

An overnight culture of strains DS-1018-3-1, DS-1071-1-1, or DS-1071-3-1were grown overnight in NZCYM media+3% dextrose+antibiotics (ampicillinat 25 μg ml⁻¹ or tetracycline at 10 μg ml⁻¹), at 37° C., with shaking. A1:40 dilution of the overnight culture was inoculated into the samemedium and grown at 37° C. with shaking until the absorbance was A₅₇₈approximately 0.3. A 1/10 volume of 10% lactose was then added to induceexpression from the T7 promoter. Cell samples were harvested about 4hours after induction by centrifuging culture samples at 5000 rpm for 10min in a Sorvall RC-3B, at 4° C.

Example 7

This Example illustrates the extraction and purification of Hin47.

Hin47 was expressed as soluble protein in E. coli. The cell pellet froma 250 ml culture, prepared as described in Example 6, was resuspended in40 ml of 50 mM Tris-HCl, pH 8.0, and disrupted by sonication (3×10 min,70% duty circle). The extract was centrifuged at 20,000×g and theresulting supernatant which contained >95% of the soluble Hin47 proteinwas retained. This fraction was called "Hin47-extract".

This Hin47-extract was further purified on a DEAE Sephacel column. Fortyml of the Hin47-extract was applied onto a 20-ml DEAE Sephacel columnequilibrated in 50 mM Tris-HCl, pH 8.0. Hin47 bound to the column underthese conditions. The column was washed with 100 ml of 50 mM Tris-HCl,pH 8.0, and then washed with 100 ml of 50 mM Tris-HCl, pH 8.0 containing20 mM NaCl. Hin47 was then eluted with 50 mM Tris-HCl, pH 8.0,containing 40 mM NaCl. The amount of Hin47 in the fractions wasdetermined by the BCA protein assay. The purity of Hin47 was assessed bySDS-PAGE analysis. The fractions containing Hin47 were combined andstored at -20° C.

Example 8

This Example illustrates the extraction and purification of Hin47Ser-197→alanine analog.

Hin47 Ser-197→alanine analog was expressed in inclusion bodies in E.coli. The cell pellet from a 250 ml culture, prepared as described inExample 6, was resuspended in 40 ml of 50 mM Tris-HCl, pH 8.0, anddisrupted by sonication (3×10 min, 70% duty circle). The extract wascentrifuged at 20,000×g and the resulting pellet was saved. The pelletwas re-extracted with 40 ml of 50 mM Tris-HCl, 0.5% Triton X-100, 10 mMEDTA, pH 8.0. The suspension was sonicated 10 min at 70% duty circle.The extract was centrifuged at 300×g for 5 min. The resultantsupernatant was centrifuged again at 20,000×g for 30 min and theresultant pellet was saved. The pellet was resuspended in 50 mMTris-HCl, 0.5% Triton X-100, 10 mM EDTA, pH 8.0. The suspension was thenmixed with 50 mM Tris-HCl, pH 8.0 containing 8 M urea. The final ureaconcentration in the mixture was adjusted to 2 M with 50 mM Tris-HCl, pH8.0. Hin47 Ser-197→alanine analog was completely solubilized under theseconditions. The final volume of the solution was 20 ml. This fraction iscalled "Hin47 analog-extract". The Hin47 analog-extract was furtherpurified on a DEAE Sephacel column. Twenty ml of Hin47 analog-extractwas applied onto a 10 ml DEAE Sephacel column equilibrated in 50 mMTris-HCl, pH 8.0. Hin47 Ser-197→alanine analog bound to the column underthese conditions. The column was washed with 50 mM Tris-HCl, pH 8.0, andHin47 analog was eluted with 50 mM Tris-HCl, pH 8.0, containing 30 mMNaCl. The amount of Hin47 analog in the fractions was determined by theBCA protein assay. The purity of Hin47 analog was assessed by SDS-PAGEanalysis (FIG. 6). The fractions containing Hin47 analog were combinedand stored at -20° C.

Example 9

This Example illustrates the protease activity of Hin47 and Hin47Ser-197→alanine analog.

The enzymatic activity of Hin47 and Hin47 Ser-197→alanine analog wasanalyzed using β-casein as a substrate (FIG. 7). The reaction mixturescontained 5 μg of βcasein and either Hin47 or Hin47 analog. The reactionwas carried out at 37° C. for two hours, and then stopped by adding theSDS-sample buffer and instantly heating the sample at 100° C. for 5 min.The aliquots were analyzed by SDS-PAGE. As shown in FIG. 7, digestion ofβ-casein by Hin47 was more obvious after two hours (panel A, lane 1) incomparison to the fractions containing Hin47 analog (panel A, lane 2) orwithout any exogenous proteins (panel A, lane 3). The presence of Hin47or Hin47 analog in these mixtures were confirmed by immuno-blottingusing a monoclonal antibody to Hin47 (FIG. 7, panel C, lanes 1 and 2).

The protease activities of Hin47 and Hin47 Ser-197→alanine analog werealso compared by analyzing the autodigestion of Hin47 or Hin47 analog at4° C. and -20° C. The purified Hin47 or analog were stored at either 4°C. or -20° C. for up to 20 days. Aliquots were taken on days 0, 10 and20 and the stability of Hin47 or analog was analyzed by immuno-blottingusing a Hin47 monoclonal antibody (FIG. 8). The analog was much morestable than Hin47 up to 20 days when stored at either 4° C. or -20° C.

To further examine the protease activity of the Hin47 Ser-197→alanineanalog, the ability of Hin47 or analog to degrade an 80-kDa H.influenzae recombinant antigen was examined. Thus, a mixed antigen studywas performed to determine the proteolytic effect of Hin47 or Hin47analog on another antigen. An 80 kDa H. influenzae recombinant protein(TBP1) was chosen for this study in order to distinguish it from theHin47 or analog protein (47 kDa). Five mixtures were formulated asfollows: 80-kDa protein alone; 80-kDa protein+Hin47; 80-kDaprotein+analog; Hin47 alone; and analog alone. The amount of eachprotein in the mixture was 5 μg. The mixtures were stored at 4° C. up tofour weeks. Aliquots were taken on days 0, 7, 14 and 28 for analysis bySDS-PAGE (FIG. 9). Both the 80 kDa protein and Hin47 were largelydegraded after one week (lanes 2 and 4). In contrast, the 80 kDa proteinin combination with Hin47 analog remained intact after one week, andshowed only slight degradation even after four weeks (lane 3).

Example 10

This Example illustrates the comparative immunogenicity of Hin47 andHin47 analog in mice. The results of a study to determine thecomparative immunogenicity of Hin47 and the Hin47 Ser-197→alanine analogare shown in FIG. 10. Thus, groups of five Balb/c mice were injectedthree times (as indicated by arrows) s.c. on days 1, 29 and 43 with 1-μgdose of either Hin47 or Hin47 analog in the presence of AlPO₄ (1.5 mgper dose). Blood samples were taken on days 14, 28, 42 and 56 (asindicated by bleeds 1, 2, 3 and 4, respectively) for analyzing theanti-Hin47 antibody titers by EIAs. The determination of anti-Hin47antibodies in mouse sera was performed as described by Panezutti et al.(1993). Microtiter wells were coated with 1 μg of either Hin47 or analogfor 16 hours at room temperature. The plates were then blocked with 0.1%(w/v) bovine serum albumin in PBS. The mouse sera were serially diluted,added to the wells, then incubated for one hour at room temperature.Affinity-purified F(ab')₂ fragments of goat anti-mouse IgG (Fc specific)antibody conjugated to horseradish peroxidase were used as the secondantibody. The reactions were developed using tetramethylbenzidine(TMB/H₂ O₂) and absorbencies were measured at 450 nm (using 540 nm as areference wavelength) in a Flow Multiskan MCC microplate reader. Thereactive titer of an antiserum was defined as the reciprocal of thedilution consistently showing a two-fold increase in absorbance overthat obtained with the pre-bleed serum sample. As can be seen from FIG.10, both Hin47 and the Hin47 analog elicited comparable IgG titers inmice regardless of whether Hin47 or mutant was used as an antigen inEIAs.

To further examine the immune response to Hin47 or the Hin47Ser-197→alanine analog, the subclasses of anti-Hin47 IgG in mouse serawere determined. Microtiter wells were coated with 1 μg of purifiedHin47 or analog. The final bleed of mouse serum samples from thecomparative immunogenicity study (as described above) were pooled andtested in EIAs. Rat anti-mouse IgG₁, IgG_(2a), IgG_(2b) antibodiesconjugated horseradish peroxidase and rabbit anti-mouse IgG₃ conjugatedto horseradish peroxidase were used as reagents in EIAs. The workingdilution of each conjugate was determined using purified antibodysubclasses to avoid cross reactivity. The reactive titers weredetermined as described above. As shown in Table 1 below, theIgG-subclass profile induced in mice by either Hin47 or Hin47 analogwere identical, regardless of whether Hin47 or analog was used as asolid antigen in the EIAs. The predominant IgG response in both groupsof mouse sera was of the IgG₁ isotype. Hence, the Hin47 analog exhibitedsubstantially the same immunogenic properties as the natural protein.

Example 11

This Example illustrates the immunoprotective properties of Hin47 andHin47 Ser-197→alanine analog.

The immunoprotective properties of Hin47 and the Hin47 Ser-197→alanineanalog were analyzed by the ability of Hin47 specific antisera toprotect infant rats against H. influenzae type b strain MinnA in abacteremia model. The results of this study are shown in Table 2 below.Groups of nine 6-day old Wistar infant rats were inoculatedsubcutaneously (s.c.) on the dorsum close to the neck with 0.1 mL ofeither a rabbit anti-Hin47 analog antiserum or the correspondingprebleed serum. Twenty-four hours later, the animals were challengedintraperitoneally (i.p.) with 700 cfu of freshly grown Hib strain MinnA.Blood samples were collected 20 hours post-challenge and plated ontochocolate agar plates. Bacterial colonies were counted after 24 hours.As shown in Table 2, three out of nine animals in the group inoculatedwith anti-Hin47 analog antiserum did not show any bacteremia in theirblood. Only one mouse in the group inoculated with anti-Hin47 analogantiserum (11%) had a higher bacteria recovery from the blood samplecompared to mice inoculated with prebleed serum. In contrast, bacteriawere recovered from all the nine mice inoculated with pre-bleed serum.Four out of nine animals (44%) in the group inoculated with pre-bleedserum showed high levels (500 to 1,000) of bacteria recovered in bloodsamples.

SUMMARY OF DISCLOSURE

In summary of this disclosure, the present invention provides a novelanalog of Haemophilus influenzae Hin47 protein which has a decreasedprotease activity of less than about 10% of that of the natural Hin47protein as well as isolated and purified DNA molecules encoding thesame.

                  TABLE 1    ______________________________________    Hin47 IgG titers in mouse immune sera           IgG titers in Group #1*                         IgG titers in Group #2*    IgG Suclass             To Hin47  To Mutant To Hin47                                         To Mutant    ______________________________________    IgG(H + L)             102,400   102,400   102,400 102,400    IgG.sub.1             25,600    25,600    25,600  25,60O    IgG.sub.2a             <100      <100      <100    <100    IgG.sub.2b             400       400       400     400    IgG.sub.3             <100      <100      <100    <100    ______________________________________     *Group #1: Immune sera were pooled from a group of five mice received     Hin47 immunization.     Group #2: Immune sera were pooled from a group of five mice received Hin4     mutant immunization.     Plates were coated with either Hin47 or mutant protein.

                  TABLE 2    ______________________________________    Protective ability of rabbit Anti-Hin47 Mutant    antiserum against Hib in infant rat model of bacteremia    Number of Animals    cfu of Bacteria/2.5 μL Blood                    Av.50    Av.200 Av.650  Total    Antibody            Av.0    (10-100) (100-300)                                    (300-1,000)                                            Animals    ______________________________________    Anti-Hin47*            3       3        2      1       9    Prebleed            0       4        1      4       9    ______________________________________

Groups of nine 6-day old infant rats were immunized s.c. with either arabbit anti-Hin47 mutant antiserum or the corresponding prebleed serum.Animals were challenged i.p. with 700 cfu H. influenzae strain MinnAafter 24 hours. The blood samples were taken at 20 hours after thechallenge.

Anti-Hin47* antibody: rabbit immune serum raised against purified Hin47mutant in CFA/IFA.

Average bacteria recovery from immunized group: 100 cfu per 2.5 μL ofblood; from control group: 290 cfu per 2.5 μL of blood.

Reference List

1. Zangwill et al, 1993 MMWR 42:1-15.

2. Schoendorf et al, 1994 Pediatrics 93:663-8.

3. Brenner et al, 1988 Nature 334:528-530.

4. O'Hagan 1992 Clin. Pharmokinet. 22:1-10.

5. Ulmer et al, 1993 Curr. Opinion. Invest. Drugs 2:983-989.

6. Chang et al, 1978 Nature 275:617.

7. Goeddel et-al 1980 Nucl. Acid. Res. 8:4057.

8. Harkness et al, 1992 J. Bacteriol. 174:2425-2430.

9. Loeb et al, 1987 Infec. Immun. 55:2612-2618.

10. Holmes and Quigley 1981. Analyt. Biochem. 114:193-197.

11. Young and Davis 1985 Gene 38:31-38.

12. Panezutti et al, 1993 Infec. Immun. 61:1867-72.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 23    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 2894 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    #ID NO:1: (xi) SEQUENCE DESCRIPTION: SEQ    - GGATCCGTTA ATACTGAAAT AAATGGCACA CCTTTTTCAC GCATTTGGGC AA - #GTACAGCA      60    - CTGGTTTTTG CCATTTGCAT TAAAGAGAAT AATGCTTCCT GCATACGAGC AC - #CACCACTC     120    - GCAGAGAAAC ATACAAACGG ACAATTCATT TCCATCGCTT TTTCAGCCGC TT - #TAACAAAT     180    - TTTGCACCAA CTACAGAACC CATTGAACCG CCCATAAAAG CAAAGTTCGA TG - #CAGCCACA     240    - ACAATTGGCA TATCATAAAG TGTACCTGTC ATAGTAATTA GCGCATCTTT CT - #CGCCCGTT     300    - TCTTTTTGTG CCGCATTGAT ACGATCTTTA TATTTCTTTA AATCTTTAAA TT - #TTAAAATA     360    - TCTTTTGGTT CTAAATCTGC CGCAATTTCT TGGCTTGAAT CTTCGTCCAA TA - #AATTTAAT     420    - AAACGCTCAC GAGCATCAAT ACGCATATGA TGACCACATT TCGGGCAAAC AT - #ACAGATTA     480    - CGTTTGAGTT CTTCACTATA AAGTACTTGT TCACAAGCAG TACATTTTGT CC - #ATACGCCT     540    - TCTGGCACAT TGGCTTTTCG AGTGGAAGAA GAAGGACTTT TACTAAAAAT TC - #GGTTAATC     600    - CAGCTCATTT TTTGACCTTT TTATTGACTA GAAAATTGCG CGTATTAGAA CA - #TAAATTTA     660    - TAGAATTTGC TACTTGTAAG ACCGTTTTTG TACTGCTCCG ATTTCCTTTT AA - #ACAAGATA     720    - ATTTGCTCTC CTCTTATTGA ACATTTTTTT TATTTTTTTG TCTTACTGAC CA - #CGTTATCT     780    - GAAATTTATT TTGGAGTATT TATGAAAAAA ACACGTTTTG TACTAAATAG TA - #TTGCACTT     840    - GGATTAAGTG TATTAAGCAC ATCATTTGTT GCTCAAGCCA CTTTGCCAAG TT - #TTGTTTCG     900    - GAACAAAACA GTCTTGCACC AATGTTAGAA AAAGTACAAC CTGCCGTTGT CA - #CTCTTTCC     960    - GTTGAAGGAA AAGCTAAAGT AGATTCTCGT TCTCCTTTCC TAGACGATAT TC - #CTGAAGAA    1020    - TTTAAATTCT TCTTTGGCGA TCGTTTTGCC GAACAATTTG GTGGACGTGG AG - #AATCAAAG    1080    - CGTAACTTCC GTGGTTTAGG TTCTGGTGTC ATTATTAATG CAAGCAAAGG CT - #ATGTTTTA    1140    - ACCAATAATC ATGTTATTGA TGAAGCTGAT AAAATTACCG TGCAATTACA AG - #ATGGGCGT    1200    - GAATTTAAAG CAAAATTAGT GGGTAAAGAT GAACTATCAG ATATTGCATT AG - #TACAGCTT    1260    - GAAAAACCAA GTAATTTAAC AGAAATCAAA TTTGCTGATT CCGACAAATT AC - #GCGTAGGC    1320    - GATTTCACTG TTGCAATCGG TAATCCATTT GGTTTAGGTC AAACTGTGAC AT - #CAGGTATT    1380    - GTTTCTGCAT TGGGTCGTTC AACAGGTTCT GACAGTGGCA CTTATGAAAA CT - #ATATTCAA    1440    - ACCGATGCAG CAGTAAACCG CGGTAATTCG GGTGGAGCGT TAGTAAACTT AA - #ATGGCGAA    1500    - CTTATTGGAA TTAATACCGC AATTATTTCT CCAAGCGGTG GCAATGCAGG AA - #TTGCCTTT    1560    - GCGATTCCAA GTAATCAAGC AAGCAATTTA GTGCAACAAA TTTTAGAATT TG - #GTCAAGTG    1620    - CGTCGCGGAT TGCTTGGTAT TAAAGGTGGC GAACTCAATG CTGATTTAGC CA - #AAGCCTTT    1680    - AATGTAAGCG CGCAACAAGG CGCATTTGTA AGTGAAGTTT TACCGAAATC TG - #CTGCTGAA    1740    - AAAGCAGGAC TTAAAGCGGG CGATATTATC ACGGCGATGA ACGGTCAAAA AA - #TCTCAAGT    1800    - TTCGCTGAAA TTCGTGCAAA AATCGCAACC ACTGGTGCAG GCAAAGAGAT TA - #GCTTGACT    1860    - TACTTACGTG ATGGCAAATC CCACGACGTT AAAATGAAAT TACAAGCGGA TG - #ATAGTAGC    1920    - CAACTTTCCT CAAAAACTGA GTTGCCTGCA TTAGATGGTG CAACATTGAA AG - #ACTACGAT    1980    - GCTAAAGGCG TTAAAGGAAT TGAAATCACA AAAATTCAAC CTAATTCGCT GG - #CTGCACAA    2040    - CGTGGTTTAA AATCGGGCGA TATTATTATT GGTATTAATC GTCAAATGAT CG - #AAAACATT    2100    - CGTGAATTAA ATAAAGTGCT TGAAACTGAA CCGTCAGCAG TTGCACTTAA TA - #TTTTACGA    2160    - GGTGACAGTA ATTTCTATTT ATTAGTGCAA TAATCTGCTT GATATATTTA AG - #AAAAAAGT    2220    - CCGATCACAA TGATCGGGCT TCTTTTTATG CAGCAATCGT TCTTAACAAA TC - #CACCACAA    2280    - ATTCTAACCG CACTTTGTTA TCAGATAAAT CTTTCATGAA CTTAAATTTT AA - #TGGGCCAT    2340    - CAAATCGATA CACAATAGGT TCTTTTTGAA TTAATTGAAT AAATTTATCT GG - #ATTCACTT    2400    - GTGCTTTTGC TGAAAACTCA ATAAAACCGC CTTGTGTTCC TGCATCAATT CG - #CACAACTT    2460    - TCAACGGCTC AACCAACAAA CGCAATTCTG CAATTTGCAG TAAATTTTTT GT - #TGCATCAG    2520    - GCAATAATCC GAATCGATCT ATTAACTCAA CTTTTAATTC ATCTAATTCT GC - #TTTACTCT    2580    - CTGCTGCAGC AATGCGTTTA TAAAAGGATA AACGCATATT CACGTCTCCT AG - #ATAATCAT    2640    - CAGGCAGTAA AGCAGGCACA CGCAATTCAA TATCCGCTTG TTGTTGCGTC AA - #TTCTTCTA    2700    - ATGATGGTTC ACGCCCTTCT TTTAACGCTT TAACCGCTGC ATCCAATAAT TC - #CATATAAA    2760    - GCGAAAAACC GATGCTTTCA ATTTGTCCAC TTTGTTCGTT TCCAAGTAAT TC - #GCCGGCAC    2820    - CACGAATCTC TAAATCGTGG GTTGCCAAGA TAAAACCAGC CCCAAGATTA TC - #AAGATTTT    2880    #   2894    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 463 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    #ID NO:2: (xi) SEQUENCE DESCRIPTION: SEQ    -      Met Lys Lys Thr Arg Phe Val Leu - # Asn Ser Ile Ala Leu Gly Leu    Ser    #   15    -      Val Leu Ser Thr Ser Phe Val Ala - # Gln Ala Thr Leu Pro Ser Phe    Val    #                 30    -      Ser Glu Gln Asn Ser Leu Ala Pro - # Met Leu Glu Lys Val Gln Pro    Ala    #             45    -      Val Val Thr Leu Ser Val Glu Gly - # Lys Ala Lys Val Asp Ser Arg    Ser    #         60    -      Pro Phe Leu Asp Asp Ile Pro Glu - # Glu Phe Lys Phe Phe Phe Gly    Asp    #     80    -      Arg Phe Ala Glu Gln Phe Gly Gly - # Arg Gly Glu Ser Lys Arg Asn    Phe    #   95    -      Arg Gly Leu Gly Ser Gly Val Ile - # Ile Asn Ala Ser Lys Gly Tyr    Val    #                110    -      Leu Thr Asn Asn His Val Ile Asp - # Glu Ala Asp Lys Ile Thr Val    Gln    #            125    -      Leu Gln Asp Gly Arg Glu Phe Lys - # Ala Lys Leu Val Gly Lys Asp    Glu    #        140    -      Leu Ser Asp Ile Ala Leu Val Gln - # Leu Glu Lys Pro Ser Asn Leu    Thr    #    160    -      Glu Ile Lys Phe Ala Asp Ser Asp - # Lys Leu Arg Val Gly Asp Phe    Thr    #   175    -      Val Ala Ile Gly Asn Pro Phe Gly - # Leu Gly Gln Thr Val Thr Ser    Gly    #                190    -      Ile Val Ser Ala Leu Gly Arg Ser - # Thr Gly Ser Asp Ser Gly Thr    Tyr    #            205    -      Glu Asn Tyr Ile Gln Thr Asp Ala - # Ala Val Asn Arg Gly Asn Ser    Gly    #        220    -      Gly Ala Leu Val Asn Leu Asn Gly - # Glu Leu Ile Gly Ile Asn Thr    Ala    #    240    -      Ile Ile Ser Pro Ser Gly Gly Asn - # Ala Gly Ile Ala Phe Ala Ile    Pro    #   255    -      Ser Asn Gln Ala Ser Asn Leu Val - # Gln Gln Ile Leu Glu Phe Gly    Gln    #                270    -      Val Arg Arg Gly Leu Leu Gly Ile - # Lys Gly Gly Glu Leu Asn Ala    Asp    #            285    -      Leu Ala Lys Ala Phe Asn Val Ser - # Ala Gln Gln Gly Ala Phe Val    Ser    #        300    -      Glu Val Leu Pro Lys Ser Ala Ala - # Glu Lys Ala Gly Leu Lys Ala    Gly    #    320    -      Asp Ile Ile Thr Ala Met Asn Gly - # Gln Lys Ile Ser Ser Phe Ala    Glu    #   335    -      Ile Arg Ala Lys Ile Ala Thr Thr - # Gly Ala Gly Lys Glu Ile Ser    Leu    #                350    -      Thr Tyr Leu Arg Asp Gly Lys Ser - # His Asp Val Lys Met Lys Leu    Gln    #            365    -      Ala Asp Asp Ser Ser Gln Leu Ser - # Ser Lys Thr Glu Leu Pro Ala    Leu    #        380    -      Asp Gly Ala Thr Leu Lys Asp Tyr - # Asp Ala Lys Gly Val Lys Gly    Ile    #    400    -      Glu Ile Thr Lys Ile Gln Pro Asn - # Ser Leu Ala Ala Gln Arg Gly    Leu    #   415    -      Lys Ser Gly Asp Ile Ile Ile Gly - # Ile Asn Arg Gln Met Ile Glu    Asn    #                430    -      Ile Arg Glu Leu Asn Lys Val Leu - # Glu Thr Glu Pro Ser Ala Val    Ala    #            445    -      Leu Asn Ile Leu Arg Gly Asp Ser - # Asn Phe Tyr Leu Leu Val Gln    #        460    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 41 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    #ID NO:3: (xi) SEQUENCE DESCRIPTION: SEQ    #   41             TTGT ATTAAATAGT ATTGCACTTG G    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 37 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    #ID NO:4: (xi) SEQUENCE DESCRIPTION: SEQ    -      Met Lys Lys Thr Arg Phe Val Leu - # Asn Ser Ile Ala Leu Gly Leu    Ser    #   15    -      Val Leu Ser Thr Ser Phe Val Ala - # Gln Ala Thr Leu Pro Ser Phe    Val    #                 30    -      Ser Glu Gln Asn Ser                 35    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 472 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    #ID NO:5: (xi) SEQUENCE DESCRIPTION: SEQ    -      Met Lys Lys Thr Thr Leu Ala Leu - # Ser Arg Leu Ala Leu Ser Leu    Ser    #   15    -      Leu Ala Leu Ser Pro Leu Ser Ala - # Thr Ala Ala Glu Thr Ser Ser    Ala    #                 30    -      Thr Thr Ala Gln Gln Met Pro Ser - # Leu Ala Pro Met Leu Glu Lys    Val    #             45    -      Met Pro Ser Val Val Ser Ile Asn - # Val Glu Gly Ser Thr Thr Val    Asn    #         60    -      Thr Pro Arg Met Pro Arg Asn Phe - # Gln Gln Phe Phe Gly Asp Asp    Ser    #     80    -      Pro Phe Cys Gln Glu Gly Ser Pro - # Phe Gln Ser Ser Pro Phe Cys    Gln    #   95    -      Gly Gly Gln Gly Gly Asn Gly Gly - # Gly Gln Gln Gln Lys Phe Met    Ala    #                110    -      Leu Gly Ser Gly Val Ile Ile Asp - # Ala Asp Lys Gly Tyr Val Val    Thr    #            125    -      Asn Asn His Val Val Asp Asn Ala - # Thr Val Ile Lys Val Gln Leu    Ser    #        140    -      Asp Gly Arg Lys Phe Asp Ala Lys - # Met Val Gly Lys Asp Pro Arg    Ser    #    160    -      Asp Ile Ala Leu Ile Gln Ile Gln - # Asn Pro Lys Asn Leu Thr Ala    Ile    #   175    -      Lys Met Ala Asp Ser Asp Ala Leu - # Arg Val Gly Asp Tyr Thr Val    Gly    #                190    -      Ile Gly Asn Pro Phe Gly Leu Gly - # Glu Thr Val Thr Ser Gly Ile    Val    #            205    -      Ser Ala Leu Gly Arg Ser Gly Leu - # Asn Ala Glu Asn Tyr Glu Asn    Phe    #        220    -      Ile Gln Thr Asp Ala Ala Ile Asn - # Arg Gly Asn Ser Gly Gly Ala    Leu    #    240    -      Val Asn Leu Asn Gly Glu Leu Ile - # Gly Ile Asn Thr Ala Ile Leu    Ala    #   255    -      Pro Asp Gly Gly Asn Ile Gly Ile - # Gly Phe Ala Ile Pro Ser Asn    Met    #                270    -      Val Lys Asn Leu Thr Ser Gln Met - # Val Glu Tyr Gly Gln Val Lys    Arg    #            285    -      Gly Glu Leu Gly Ile Met Gly Thr - # Glu Leu Asn Ser Glu Leu Ala    Lys    #        300    -      Ala Met Lys Val Asp Ala Gln Arg - # Gly Ala Phe Val Ser Gln Val    Leu    #    320    -      Pro Asn Ser Ser Ala Ala Lys Ala - # Gly Ile Lys Ala Gly Asp Val    Ile    #   335    -      Thr Ser Leu Asn Gly Lys Pro Ile - # Ser Ser Phe Ala Ala Leu Arg    Ala    #                350    -      Gln Val Gly Thr Met Pro Val Gly - # Ser Lys Leu Thr Leu Gly Leu    Leu    #            365    -      Arg Asp Gly Lys Gln Val Asn Val - # Asn Leu Glu Leu Gln Gln Ser    Ser    #        380    -      Gln Asn Gln Val Asp Ser Ser Ser - # Ile Phe Asn Gly Ile Glu Gly    Ala    #    400    -      Glu Met Ser Asn Lys Gly Lys Asp - # Gln Gly Val Val Val Asn Asn    Val    #   415    -      Lys Thr Gly Thr Pro Ala Ala Gln - # Ile Gly Leu Lys Lys Gly Asp    Val    #                430    -      Ile Ile Gly Ala Asn Gln Ile Ala - # Val Lys Asn Ile Ala Glu Ile    Arg    #            445    -      Lys Val Leu Asp Ser Lys Pro Ser - # Val Leu Ala Leu Asn Ile Gln    Arg    #        460    -      Gly Asp Arg His Leu Pro Val Asn    #    470    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 475 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    #ID NO:6: (xi) SEQUENCE DESCRIPTION: SEQ    -      Met Lys Lys Thr Thr Leu Ala Met - # Ser Ala Leu Ala Leu Ser Leu    Gly    #   15    -      Leu Ala Leu Ser Pro Leu Ser Ala - # Thr Ala Ala Glu Thr Ser Ser    Ser    #                 30    -      Ala Met Thr Ala Gln Gln Met Pro - # Ser Leu Ala Pro Met Leu Glu    Lys    #             45    -      Val Met Pro Ser Val Val Ser Ile - # Asn Val Glu Gly Ser Thr Thr    Val    #         60    -      Asn Thr Pro Arg Met Pro Arg Asn - # Phe Gln Gln Phe Phe Gly Asp    Asp    #     80    -      Ser Pro Phe Cys Gln Asp Gly Ser - # Pro Phe Gln Asn Ser Pro Phe    Cys    #   95    -      Gln Gly Gly Gly Asn Gly Gly Asn - # Gly Gly Gln Gln Gln Lys Phe    Met    #                110    -      Ala Leu Gly Ser Gly Val Ile Ile - # Asp Ala Asp Lys Gly Tyr Val    Val    #            125    -      Thr Asn Asn His Val Val Asp Asn - # Ala Ser Val Ile Lys Val Gln    Leu    #        140    -      Ser Asp Gly Arg Lys Phe Asp Ala - # Lys Val Val Gly Lys Asp Pro    Arg    #    160    -      Ser Asp Ile Ala Leu Ile Gln Ile - # Gln Asn Pro Lys Asn Leu Thr    Ala    #   175    -      Ile Lys Leu Ala Asp Ser Asp Ala - # Leu Arg Val Gly Asp Tyr Thr    Val    #                190    -      Ala Ile Gly Asn Pro Phe Gly Leu - # Gly Glu Thr Val Thr Ser Gly    Ile    #            205    -      Val Ser Ala Leu Gly Arg Ser Gly - # Leu Asn Val Glu Asn Tyr Glu    Asn    #        220    -      Phe Ile Gln Thr Asp Ala Ala Ile - # Asn Arg Gly Asn Ser Gly Gly    Ala    #    240    -      Leu Val Asn Leu Asn Gly Glu Leu - # Ile Gly Ile Asn Thr Ala Ile    Leu    #   255    -      Ala Pro Asp Gly Gly Asn Ile Gly - # Ile Gly Phe Ala Ile Pro Ser    Asn    #                270    -      Met Val Lys Asn Leu Thr Ser Gln - # Met Val Glu Tyr Gly Gln Val    Arg    #            285    -      Arg Gly Glu Leu Gly Ile Met Gly - # Thr Glu Leu Asn Ser Glu Leu    Ala    #        300    -      Lys Ala Met Lys Val Asp Ala Gln - # Arg Gly Ala Phe Val Ser Gln    Val    #    320    -      Met Pro Asn Ser Ser Ala Ala Lys - # Ala Gly Ile Lys Ala Gly Asp    Val    #   335    -      Ile Thr Ser Leu Asn Gly Lys Pro - # Ile Ser Ser Phe Ala Ala Leu    Arg    #                350    -      Ala Gln Val Gly Thr Met Pro Val - # Gly Ser Lys Ile Ser Leu Gly    Leu    #            365    -      Leu Arg Glu Gly Lys Ala Ile Thr - # Val Asn Leu Glu Leu Gln Gln    Ser    #        380    -      Ser Gln Ser Gln Val Asp Ser Ser - # Thr Ile Phe Ser Gly Ile Glu    Gly    #    400    -      Ala Glu Met Ser Asn Lys Gly Gln - # Asp Lys Gly Val Val Val Ser    Ser    #   415    -      Val Lys Ala Asn Ser Pro Ala Ala - # Gln Ile Gly Leu Lys Lys Gly    Asp    #                430    -      Val Ile Ile Gly Ala Asn Gln Ile - # Pro Val Lys Asn Ile Ala Glu    Ile    #            445    -      Arg Lys Ile Leu Asp Ser Lys Pro - # Ser Val Leu Ala Leu Asn Ile    Gln    #        460    -      Arg Gly Asp Ser Ser Ile Tyr Leu - # Leu Met Gln    #    475    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 228 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    #ID NO:7: (xi) SEQUENCE DESCRIPTION: SEQ    -      Ile Val Gly Gly Tyr Lys Cys Glu - # Lys Asn Ser Gln Pro Trp Gln    Val    #   15    -      Ala Val Ile Asn Glu Tyr Leu Cys - # Gly Gly Val Leu Ile Asp Pro    Ser    #                 30    -      Trp Val Ile Thr Ala Ala His Cys - # Tyr Ser Asn Asn Tyr Gln Val    Leu    #             45    -      Leu Gly Arg Asn Asn Leu Phe Lys - # Asp Glu Pro Phe Ala Gln Arg    Arg    #         60    -      Leu Val Pro Gln Ser Phe Arg His - # Pro Asp Tyr Ile Pro Leu Ile    Pro    #     80    -      Val His Asp His Ser Asn Asp Leu - # Met Leu Leu His Leu Ser Glu    Pro    #   95    -      Ala Asp Ile Thr Gly Gly Val Lys - # Val Ile Asp Leu Pro Thr Lys    Glu    #                110    -      Pro Lys Val Gly Ser Thr Cys Leu - # Ala Ser Gly Trp Gly Ser Thr    Asn    #            125    -      Pro Ser Glu Met Val Val Ser His - # Asp Leu Gln Cys Val Asn Ile    His    #        140    -      Leu Leu Ser Asn Glu Lys Cys Ile - # Glu Thr Tyr Lys Asp Asn Val    Thr    #    160    -      Asp Val Met Leu Cys Ala Gly Glu - # Met Glu Gly Gly Lys Asp Thr    Cys    #   175    -      Ala Gly Asp Ser Gly Gly Pro Leu - # Ile Cys Asp Gly Val Leu Gln    Gly    #                190    -      Ile Thr Ser Gly Gly Ala Thr Pro - # Cys Ala Lys Pro Lys Thr Pro    Ala    #            205    -      Ile Tyr Ala Lys Leu Ile Lys Phe - # Thr Ser Trp Ile Lys Lys Val    Met    #        220    -      Lys Glu Asn Pro         225    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 232 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    #ID NO:8: (xi) SEQUENCE DESCRIPTION: SEQ    -      Ile Ile Gly Gly Arg Glu Cys Glu - # Lys Asn Ser His Pro Trp Gln    Val    #   15    -      Ala Ile Tyr His Tyr Ser Ser Phe - # Gln Cys Gly Gly Val Leu Val    Asn    #                 30    -      Pro Lys Trp Val Leu Thr Ala Ala - # His Cys Lys Asn Asp Asn Tyr    Glu    #             45    -      Val Trp Leu Gly Arg His Asn Leu - # Phe Glu Asn Glu Asn Thr Ala    Gln    #         60    -      Phe Phe Gly Val Thr Ala Asp Phe - # Pro His Pro Gly Phe Asn Leu    Ser    #     80    -      Ala Asp Gly Lys Asp Tyr Ser His - # Asp Leu Met Leu Leu Arg Leu    Gln    #   95    -      Ser Pro Ala Lys Ile Thr Asp Ala - # Val Lys Val Leu Glu Leu Pro    Thr    #                110    -      Gln Glu Pro Glu Leu Gly Ser Thr - # Cys Glu Ala Ser Gly Trp Gly    Ser    #            125    -      Ile Glu Pro Gly Pro Asp Asp Phe - # Glu Phe Pro Asp Glu Ile Gln    Cys    #        140    -      Val Gln Leu Thr Leu Leu Gln Asn - # Thr Phe Cys Ala Asp Ala His    Pro    #    160    -      Asp Lys Val Thr Glu Ser Met Leu - # Cys Ala Gly Tyr Leu Pro Gly    Gly    #   175    -      Lys Asp Thr Cys Met Gly Asp Ser - # Gly Gly Pro Leu Ile Cys Asn    Gly    #                190    -      Met Trp Gln Gly Ile Thr Ser Trp - # Gly His Thr Pro Cys Gly Ser    Ala    #            205    -      Asn Lys Pro Ser Ile Tyr Thr Lys - # Leu Ile Phe Tyr Leu Asp Trp    Ile    #        220    -      Asp Asp Thr Ile Thr Glu Asn Pro    #    230    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 223 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    #ID NO:9: (xi) SEQUENCE DESCRIPTION: SEQ    -      Ile Val Gly Gly Tyr Thr Cys Gly - # Ala Asn Thr Val Pro Tyr Gln    Val    #   15    -      Ser Leu Asn Ser Gly Tyr His Phe - # Cys Gly Gly Ser Leu Ile Asn    Ser    #                 30    -      Gln Trp Val Val Ser Ala Ala His - # Cys Tyr Lys Ser Gly Ile Gln    Val    #             45    -      Arg Leu Gly Glu Asp Asn Ile Asn - # Val Val Glu Gly Asn Glu Gln    Phe    #         60    -      Ile Ser Ala Ser Lys Ser Ile Val - # His Pro Ser Tyr Asn Ser Asn    Thr    #     80    -      Leu Asn Asn Asp Ile Met Leu Ile - # Lys Leu Lys Ser Ala Ala Ser    Leu    #   95    -      Asn Ser Arg Val Ala Ser Ile Ser - # Leu Pro Thr Ser Cys Ala Ser    Ala    #                110    -      Gly Thr Gln Cys Leu Ile Ser Gly - # Trp Gly Asn Thr Lys Ser Ser    Gly    #            125    -      Thr Ser Tyr Pro Asp Val Leu Lys - # Cys Leu Lys Ala Pro Ile Leu    Ser    #        140    -      Asp Ser Ser Cys Lys Ser Ala Tyr - # Pro Gly Gln Ile Thr Ser Asn    Met    #    160    -      Phe Cys Ala Gly Tyr Leu Glu Gly - # Gly Lys Asp Ser Cys Gln Gly    Asp    #   175    -      Ser Gly Gly Pro Val Val Cys Ser - # Gly Lys Leu Gln Gly Ile Val    Ser    #                190    -      Trp Gly Ser Gly Cys Ala Gln Lys - # Asn Lys Pro Gly Val Tyr Thr    Lys    #            205    -      Val Cys Asn Tyr Val Ser Trp Ile - # Lys Gln Thr Ile Ala Ser Asn    #        220    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 228 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    #ID NO:10:(xi) SEQUENCE DESCRIPTION: SEQ    -      Ile Val Asn Gly Glu Glu Ala Val - # Pro Gly Ser Trp Pro Trp Gln    Val    #   15    -      Ser Leu Gln Asp Lys Thr Gly Phe - # His Phe Cys Gly Gly Ser Leu    Ile    #                 30    -      Asn Glu Asn Trp Val Val Thr Ala - # Ala His Cys Gly Val Thr Thr    Ser    #             45    -      Asp Val Val Val Ala Gly Glu Phe - # Asp Gln Gly Ser Ser Ser Glu    Lys    #         60    -      Ile Gln Lys Leu Lys Ile Ala Lys - # Val Phe Lys Asn Ser Lys Tyr    Asn    #     80    -      Ser Leu Thr Ile Asn Asn Asp Ile - # Thr Leu Leu Lys Leu Ser Thr    Ala    #   95    -      Ala Ser Phe Ser Gln Thr Val Ser - # Ala Val Cys Leu Pro Ser Ala    Ser    #                110    -      Asp Asp Phe Ala Ala Gly Thr Thr - # Cys Val Thr Thr Gly Trp Gly    Leu    #            125    -      Thr Arg Tyr Ala Asn Thr Pro Asp - # Arg Leu Gln Gln Ala Ser Leu    Pro    #        140    -      Leu Leu Ser Asn Thr Asn Cys Lys - # Lys Tyr Trp Gly Thr Lys Ile    Lys    #    160    -      Asp Ala Met Ile Cys Ala Gly Ala - # Ser Gly Val Ser Ser Cys Met    Gly    #   175    -      Asp Ser Gly Gly Pro Leu Val Cys - # Lys Lys Asn Gly Ala Trp Thr    Leu    #                190    -      Val Gly Ile Val Ser Trp Gly Ser - # Ser Thr Cys Ser Thr Ser Thr    Pro    #            205    -      Gly Val Tyr Ala Arg Val Thr Ala - # Leu Val Asn Trp Val Gln Gln    Thr    #        220    -      Leu Ala Ala Asn         225    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 240 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    #ID NO:11:(xi) SEQUENCE DESCRIPTION: SEQ    -      Val Val Gly Gly Thr Glu Ala Gln - # Arg Asn Ser Trp Pro Ser Gln    Ile    #   15    -      Ser Leu Gln Tyr Arg Ser Gly Ser - # Ser Trp Ala His Thr Cys Gly    Gly    #                 30    -      Thr Leu Ile Arg Gln Asn Trp Val - # Met Thr Ala Ala His Cys Val    Asp    #             45    -      Arg Glu Leu Thr Phe Arg Val Val - # Val Gly Glu His Asn Leu Asn    Gln    #         60    -      Asn Asn Gly Thr Glu Gln Tyr Val - # Gly Val Gln Lys Ile Val Val    His    #     80    -      Pro Tyr Trp Asn Thr Asp Asp Val - # Ala Ala Gly Tyr Asp Ile Ala    Leu    #   95    -      Leu Arg Leu Ala Gln Ser Val Thr - # Leu Asn Ser Tyr Val Gln Leu    Gly    #                110    -      Val Leu Pro Arg Ala Gly Thr Ile - # Leu Ala Asn Asn Ser Pro Cys    Tyr    #            125    -      Ile Thr Gly Trp Gly Leu Thr Arg - # Thr Asn Gly Gln Leu Ala Gln    Thr    #        140    -      Leu Gln Gln Ala Tyr Leu Pro Thr - # Val Asp Tyr Ala Ile Cys Ser    Ser    #    160    -      Ser Ser Tyr Trp Gly Ser Thr Val - # Lys Asn Ser Met Val Cys Ala    Gly    #   175    -      Gly Asp Gly Val Arg Ser Gly Cys - # Gln Gly Asp Ser Gly Gly Pro    Leu    #                190    -      His Cys Leu Val Asn Gly Gln Tyr - # Ala Val His Gly Val Thr Ser    Phe    #            205    -      Val Ser Arg Leu Gly Cys Asn Val - # Thr Arg Lys Pro Thr Val Phe    Thr    #        220    -      Arg Val Ser Ala Tyr Ile Ser Trp - # Ile Asn Asn Val Ile Ala Ser    Asn    #    240    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 224 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    #ID NO:12:(xi) SEQUENCE DESCRIPTION: SEQ    -      Ile Ile Gly Gly Val Glu Ser Ile - # Pro His Ser Arg Pro Tyr Met    Ala    #   15    -      His Leu Asp Ile Val Thr Glu Lys - # Gly Leu Arg Val Ile Cys Gly    Gly    #                 30    -      Phe Leu Ile Ser Arg Gln Phe Val - # Leu Thr Ala Ala His Cys Lys    Gly    #             45    -      Arg Glu Ile Thr Val Ile Leu Gly - # Ala His Asp Val Arg Lys Arg    Glu    #         60    -      Ser Thr Gln Gln Lys Ile Lys Val - # Glu Lys Gln Ile Ile His Glu    Ser    #     80    -      Tyr Asn Ser Val Pro Asn Leu His - # Asp Ile Met Leu Leu Lys Leu    Glu    #   95    -      Lys Lys Val Glu Leu Thr Pro Ala - # Val Asn Val Val Pro Leu Pro    Ser    #                110    -      Pro Ser Asp Phe Ile His Pro Gly - # Ala Met Cys Trp Ala Ala Gly    Trp    #            125    -      Gly Lys Thr Gly Val Arg Asp Pro - # Thr Ser Tyr Thr Leu Arg Glu    Val    #        140    -      Glu Leu Arg Ile Met Asp Glu Lys - # Ala Cys Val Asp Tyr Arg Tyr    Tyr    #    160    -      Glu Tyr Lys Phe Gln Val Cys Val - # Gly Ser Pro Thr Thr Leu Arg    Ala    #   175    -      Ala Phe Met Gly Asp Ser Gly Gly - # Pro Leu Leu Cys Ala Gly Val    Ala    #                190    -      His Gly Ile Val Ser Tyr Gly His - # Pro Asp Ala Lys Pro Pro Ala    Ile    #            205    -      Phe Thr Arg Val Ser Thr Tyr Val - # Pro Trp Ile Asn Ala Val Ile    Asn    #        220    - (2) INFORMATION FOR SEQ ID NO:13:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 223 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    #ID NO:13:(xi) SEQUENCE DESCRIPTION: SEQ    -      Val Val Gly Gly Thr Arg Ala Ala - # Gln Gly Glu Phe Pro Phe Met    Val    #   15    -      Arg Leu Ser Met Gly Cys Gly Gly - # Ala Leu Tyr Ala Gln Asp Ile    Val    #                 30    -      Leu Thr Ala Ala His Cys Val Ser - # Gly Ser Gly Asn Asn Thr Ser    Ile    #             45    -      Thr Ala Thr Gly Gly Val Val Asp - # Leu Gln Ser Gly Ala Ala Val    Lys    #         60    -      Val Arg Ser Thr Lys Val Leu Gln - # Ala Pro Gly Tyr Asn Gly Thr    Gly    #     80    -      Lys Asp Trp Ala Leu Ile Lys Leu - # Ala Gln Pro Ile Asn Gln Pro    Thr    #   95    -      Leu Lys Ile Ala Thr Thr Thr Ala - # Tyr Asn Gln Gly Thr Phe Thr    Val    #                110    -      Ala Gly Trp Gly Ala Asn Arg Glu - # Gly Gly Ser Gln Gln Arg Tyr    Leu    #            125    -      Leu Lys Ala Asn Val Pro Phe Val - # Ser Asp Ala Ala Cys Arg Ser    Ala    #        140    -      Tyr Gly Asn Glu Leu Val Ala Asn - # Glu Glu Ile Cys Ala Gly Tyr    Pro    #    160    -      Asp Thr Gly Gly Val Asp Thr Cys - # Gln Gly Asp Ser Gly Gly Pro    Met    #   175    -      Phe Arg Lys Asp Asn Ala Asp Glu - # Trp Ile Gln Val Gly Ile Val    Ser    #                190    -      Trp Gly Tyr Gly Cys Ala Arg Pro - # Gly Tyr Pro Gly Val Tyr Thr    Glu    #            205    -      Val Ser Thr Phe Ala Ser Ala Ile - # Ala Ser Ala Ala Arg Thr Leu    #        220    - (2) INFORMATION FOR SEQ ID NO:14:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 185 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    #ID NO:14:(xi) SEQUENCE DESCRIPTION: SEQ    -      Ile Ser Gly Gly Asp Ala Ile Tyr - # Ser Ser Thr Gly Arg Cys Ser    Leu    #   15    -      Gly Phe Asn Val Arg Ser Gly Ser - # Thr Tyr Tyr Phe Leu Thr Ala    Gly    #                 30    -      His Cys Thr Asp Gly Ala Thr Thr - # Trp Trp Ala Asn Ser Ala Arg    Thr    #             45    -      Thr Val Leu Gly Thr Thr Ser Gly - # Ser Ser Phe Pro Asn Asn Asp    Tyr    #         60    -      Gly Ile Val Arg Tyr Thr Asn Thr - # Thr Ile Pro Lys Asp Gly Thr    Val    #     80    -      Gly Gly Gln Asp Ile Thr Ser Ala - # Ala Asn Ala Thr Val Gly Met    Ala    #   95    -      Val Thr Arg Arg Gly Ser Thr Thr - # Gly Thr His Ser Gly Ser Val    Thr    #                110    -      Ala Leu Asn Ala Thr Val Asn Tyr - # Gly Gly Gly Asp Val Val Tyr    Gly    #            125    -      Met Ile Arg Thr Asn Val Cys Ala - # Glu Pro Gly Asp Ser Gly Gly    Pro    #        140    -      Leu Tyr Ser Gly Thr Arg Ala Ile - # Gly Leu Thr Ser Gly Gly Ser    Gly    #    160    -      Asn Cys Ser Ser Gly Gly Thr Thr - # Phe Phe Gln Pro Val Thr Glu    Ala    #   175    -      Leu Val Ala Tyr Gly Val Ser Val - # Tyr    #                185    - (2) INFORMATION FOR SEQ ID NO:15:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 181 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    #ID NO:15:(xi) SEQUENCE DESCRIPTION: SEQ    -      Ile Ala Gly Gly Glu Ala Ile Thr - # Thr Gly Gly Ser Arg Cys Ser    Leu    #   15    -      Gly Phe Asn Val Ser Val Asn Gly - # Val Ala His Ala Leu Thr Ala    Gly    #                 30    -      His Cys Thr Asn Ile Ser Ala Ser - # Trp Ser Ile Gly Thr Arg Thr    Gly    #             45    -      Thr Ser Phe Pro Asn Asn Asp Tyr - # Gly Ile Ile Arg His Ser Asn    Pro    #         60    -      Ala Ala Ala Asp Gly Arg Val Tyr - # Leu Tyr Asn Gly Ser Tyr Gln    Asp    #     80    -      Ile Thr Thr Ala Gly Asn Ala Phe - # Val Gly Gln Ala Val Gln Arg    Ser    #   95    -      Gly Ser Thr Thr Gly Leu Arg Ser - # Gly Ser Val Thr Gly Leu Asn    Ala    #                110    -      Thr Val Asn Tyr Gly Ser Ser Gly - # Ile Val Tyr Gly Met Ile Gln    Thr    #            125    -      Asn Val Cys Ala Gln Pro Gly Asp - # Ser Gly Gly Ser Leu Phe Ala    Gly    #        140    -      Ser Thr Ala Leu Gly Leu Thr Ser - # Gly Gly Ser Gly Asn Cys Arg    Thr    #    160    -      Gly Gly Thr Thr Phe Tyr Gln Pro - # Val Thr Glu Ala Leu Ser Ala    Tyr    #   175    -      Gly Ala Thr Val Leu                     180    - (2) INFORMATION FOR SEQ ID NO:16:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 198 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    #ID NO:16:(xi) SEQUENCE DESCRIPTION: SEQ    -      Ala Asn Ile Val Gly Gly Ile Glu - # Tyr Ser Ile Asn Asn Ala Ser    Leu    #   15    -      Cys Ser Val Gly Phe Ser Val Thr - # Arg Gly Ala Thr Lys Gly Phe    Val    #                 30    -      Thr Ala Gly His Cys Gly Thr Val - # Asn Ala Thr Ala Arg Ile Gly    Gly    #             45    -      Ala Val Val Gly Thr Phe Ala Ala - # Arg Val Phe Pro Gly Asn Asp    Arg    #         60    -      Ala Trp Val Ser Leu Thr Ser Ala - # Gln Thr Leu Leu Pro Arg Val    Ala    #     80    -      Asn Gly Ser Ser Phe Val Thr Val - # Arg Gly Ser Thr Glu Ala Ala    Val    #   95    -      Gly Ala Ala Val Cys Arg Ser Gly - # Arg Thr Thr Gly Tyr Gln Cys    Gly    #                110    -      Thr Ile Thr Ala Lys His Val Thr - # Ala Asn Tyr Ala Glu Gly Ala    Val    #            125    -      Arg Gly Leu Thr Gln Gly Asn Ala - # Cys Met Gly Arg Gly Asp Ser    Gly    #        140    -      Gly Ser Trp Ile Thr Ser Ala Gly - # Gln Ala Gln Gly Val Met Ser    Gly    #    160    -      Gly Asn Val Gln Ser Asn Gly Asn - # Asn Cys Gly Ile Pro Ala Ser    Gln    #   175    -      Arg Ser Ser Leu Phe Glu Arg Leu - # Gln Pro Ile Leu Ser Gln Tyr    Gly    #                190    -      Leu Ser Leu Val Thr Gly                 195    - (2) INFORMATION FOR SEQ ID NO:17:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 7 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    #ID NO:17:(xi) SEQUENCE DESCRIPTION: SEQ    -      Gly Asn Ser Gly Gly Ala Leu    #  5 1    - (2) INFORMATION FOR SEQ ID NO:18:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 7 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    #ID NO:18:(xi) SEQUENCE DESCRIPTION: SEQ    -      Gly Asp Ser Gly Gly Pro Lys    #  5 1    - (2) INFORMATION FOR SEQ ID NO:19:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 14 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    #ID NO:19:(xi) SEQUENCE DESCRIPTION: SEQ    -      Met Lys Lys Thr Arg Phe Val Leu - # Asn Ser Ile Ala Leu Gly    #   10    - (2) INFORMATION FOR SEQ ID NO:20:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 22 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    #ID NO:20:(xi) SEQUENCE DESCRIPTION: SEQ    #                 22CGC GG    - (2) INFORMATION FOR SEQ ID NO:21:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 21 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    #ID NO:21:(xi) SEQUENCE DESCRIPTION: SEQ    #21                TTGG T    - (2) INFORMATION FOR SEQ ID NO:22:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 21 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    #ID NO:22:(xi) SEQUENCE DESCRIPTION: SEQ    #21                AGTT C    - (2) INFORMATION FOR SEQ ID NO:23:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 11 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    #ID NO:23:(xi) SEQUENCE DESCRIPTION: SEQ    -      Lys Phe Phe Phe Gly Asp Arg Phe - # Ala Glu Gln    #   10    __________________________________________________________________________

What we claim is:
 1. An isolated and purified analog of Haemophilusinfluenzae Hin47 protein having a decreased protease activity which isless than about 10% of that of natural Hin47 protein.
 2. The analog ofclaim 1 having substantially the same immunogenic properties as naturalHin47 protein.
 3. The analog of claim 1 wherein at least one amino acidof the natural Hin47 protein contributing to protease activity has beendeleted or replaced by a different amino acid, or at least one aminoacid has been inserted into the natural Hin47 protein, to provide saidreduced protease activity.
 4. The analog of claim 3 wherein said atleast one deleted or replaced amino acid is selected from amino acids195 to 201 of natural Hin47 protein.
 5. The analog of claim 4 whereinsaid at least one amino acid is Serine-197.
 6. The analog of claim 5wherein Serine-197 is replaced by alanine.
 7. The analog of claim 3wherein said at least one amino acid is Histidine-91 or Asp-121 ofnatural Hin47 protein.
 8. The analog of claim 7 wherein Histidine-91 isreplaced by alanine, lysine or arginine.
 9. The analog of claim 7wherein Asp-121 is replaced by alanine or glutamic acid.
 10. An isolatedand purified nucleic acid molecule comprising a mutant Haemophilusinfluenzae hin47 gene encoding an analog of Haemophilus influenzae Hin47protein having a decreased protease activity which is less than about10% of that of natural Hin47 protein.
 11. The nucleic acid molecule ofclaim 10 wherein said encoded analog has substantially the immunogenicproperties of natural Hin47 protein.
 12. The nucleic acid molecule ofclaim 10 wherein at least one codon of a wild-type hin47 gene encodingan amino acid contributing to protease activity has been deleted orreplaced, or at least one codon has been inserted into the wild-typehin47 gene to form said mutant hin47 gene.
 13. The nucleic acid moleculeof claim 12 wherein the at least one deleted or replaced codon encodesat least one amino acid from amino acids 195 to 201 of natural Hin47protein.
 14. The nucleic acid molecule of claim 13 wherein the at leastone codon is that encoding Serine-197.
 15. The nucleic acid molecule ofclaim 14 wherein the codon encoding Serine-197 is replaced by a codonencoding alanine.
 16. The nucleic acid molecule of claim 12 wherein theat least one codon encodes His-91 or Asp-121 of natural Hin47 protein.17. The nucleic acid molecule of claim 16 wherein the codon encodingHis-91 is replaced by a codon encoding alanine, lysine or arginine. 18.The nucleic acid molecule of claim 16 wherein the codon encoding Asp-121is replaced by a codon encoding alanine or glutamic acid.
 19. Thenucleic acid molecule of claim 10 wherein said mutant gene is formed bysite-directed mutagenesis of a wild-type hin47 gene.
 20. A recombinantplasmid adapted for transformation of a host comprising a plasmid vectorinto which has been inserted the nucleic acid molecule of claim
 10. 21.The recombinant plasmid of claim 20 which is plasmid DS-1011-1-1(pT7/Hin47*) deposited under ATCC designation no.
 75845. 22. Atransformed cell containing the recombinant plasmid of claim
 20. 23. Amethod for producing an analog of Haemophilus influenzae Hin47 proteinhaving a reduced protease activity which is less than about 10% ofnatural Hin47 protein, which comprises:identifying at least one aminoacid residue of Hin47 protein which contributes to protease activitythereof; effecting site-directed mutagenesis of the hin47 gene to removeor replace a nucleotide sequence encoding said at least one amino acidand to produce a mutated hin47 gene; introducing the mutated hin47 geneinto a cell to produce a transformed cell; and growing the transformedcell to produce the Hin47 analog.
 24. The method of claim 23 whereinsaid at least one amino acid is selected from amino acids 95 to 201 ofnatural Hin47 protein.
 25. The method of claim 24 wherein said at leastone amino acid is Serine-197.
 26. The method of claim 24 whereinSerine-197 is replaced by alanine.
 27. The method of claim 23 whereinsaid at least one amino acid is Histidine-91 or Asp-121 of natural Hin47protein.
 28. The method of claim 27 wherein Histidine-91 is replaced byalanine, lysine or arginine.
 29. The method of claim 27 wherein Asp-121is replaced by alanine or glutamic acid.
 30. The method of claim 23wherein said introduction of the mutated hin47 gene produces atransformed cell in which the mutated hin47 gene is under control of theT7 promoter, and said growing of said transformed cell and expression ofthe Hin47 analog by said T7 promoter is effected by culturing in aninducing concentration of lactose.
 31. The method of claim 30 whereinsaid introduction of the mutated hin47 gene is effected by transformingsaid cell with the recombinant plasmid DS-1011-1-1 (pT7/Hin47*)deposited under ATCC designation
 75845. 32. A method of providing anisolated and purified analog of Haemophilus influenzae Hin47 having areduced protease activity which is less than about 10% of natural Hin47protein, which comprises:identifying at least one amino acid residue ofHin47 protein which contributes to protease activity thereof; effectingsite-directed mutagenesis of the hin47 gene to remove or replace anucleotide sequence encoding said at least one amino acid and to producea mutated hin47 gene; introducing the mutated hin47 gene into a cell toproduce a transformed cell; growing the transformed cell to producegrown transformed cells harbouring inclusion bodies containing the Hin47analog; disrupting said grown transformed cells to produce supernatantand said inclusion bodies; solubilizing said inclusion bodies to producea solution containing Hin47 analog; chromatographically purifying saidHin47 analog from said solution free from cell debris; and isolatingsaid purified Hin47 analog.
 33. The method of claim 32 wherein saidintroduction of mutated hin47 gene produces a transformed cell in whichthe mutated hin47 gene is under control of the T7 promoter, and saidgrowing of said transformed cell and dispersion of the Hin47 analog bysaid T7 promoter is effected by culturing in an inducing concentrationof lactose.
 34. A chimeric molecule, comprising an analog as claimed inclaim 1 linked to a polypeptide, protein or a polysaccharide.
 35. Themethod of claim 32 wherein said at least one amino acid is selected fromamino acids 95 to 201 of natural Hin47 protein.
 36. The method of claim35 wherein said at least one amino acid is Serine-197.
 37. The method ofclaim 35 wherein Serine-197 is replaced by alanine.
 38. The method ofclaim 32 wherein said at least one amino acid is Histidine-91 or Asp-121of natural Hin47 protein.
 39. The method of claim 38 whereinHistidine-91 is replaced by alanine, lysine or arginine.
 40. The methodof claim 38 wherein Asp-121 is replaced by alanine or glutamic acid.